Activators of human pyruvate kinase

ABSTRACT

Disclosed are pyruvate kinase M2 activators, which are bis sulfonamide piperazinyl and piperidinyl compounds of Formula (I), 2,4-disubstituted 4H-thieno[3,2-c]pyrrole-2-(substituted benzyl)pyridazin-3(2H)-ones of Formula (II) and 6-(3,4-dimethylphenylaminosulfonyl)-3,4-dihydro-1H-quinolin-2-one of formula (III), wherein L, R 1 , R 2 , R 11  to R 16 , R 21  and R 22  are as defined herein, that are useful in treating a number of diseases that are treatable by the activation of PKM2, for example, cancer and anemia.

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/104,091, filed Oct. 9, 2008, the disclosure of whichis incorporated by reference.

BACKGROUND OF THE INVENTION

Pyruvate kinase (PK) is a critical metabolic enzyme operating at theultimate step in glycolysis where it catalyzes the transfer of aphosphate group from phosphoenolpyruvate to adenosine diphosphate (ADP),yielding one molecule of pyruvate and one molecule of adenosinetriphosphate (ATP). In humans there are two pyruvate kinase genes andeach produces two distinct gene products by alternative splicing. The Lgene produces two different mRNAs that differ only in the first exon toproduce the L (liver specific) and R (red blood cell) specific isozymes.Splicing of a single exon within the M gene produces the M1 isozyme thatis found in most adult tissues and the M2 isozyme that is present infetal tissues and is found to be re-expressed in tumors. Therefore,after embryonic development, adult tissues switch to either expressPK-M1 or the tissue specific L or R isozymes. However, in all tumors orcell lines of cancer lineage (including those typically expressingeither the L or R isozymes), PK gene expression reverts entirely to theM2 isoform.

PK is a tetrameric enzyme composed of four identical monomers that forma dimer of dimers in the final tetrameric structure. In humans, the M2,L, and R isozymes are activated by fructose-1,6-bis phosphate (FBP) thatbinds to a flexible loop region at the interface of the two dimers.Activation of PK shifts the enzyme to a state showing high affinity forphosphoenolpyruvate (PEP). In contrast, the M1 isoform is not regulatedby FBP and displays only high affinity PEP binding similar to theactivated state of PK.

Tumor cells undergo a metabolic transformation that is required tosupply the biochemical precursors necessary for rapid cell growth andproliferation.

Various phosphotyrosine peptides can bind to PK-M2 near the activationloop that results in the removal of FBP from the enzyme whicheffectively down-regulates PK-M2 activity. When PK-M2 is activated,glucose is converted to pyruvate. However, when PK-M2 is inactivated, abuild-up of glycolytic intermediates occurs which intermediates can bediverted towards nucleotide and lipid biosynthesis required for cellgrowth and proliferation.

In addition, PK deficiency is the second most common cause ofenzyme-deficient hemolytic anemia, following G6PD deficiency. Inpatients with PK deficiency, a metabolic block is created in the pathwayat the level of the deficient enzyme. Intermediate by-products andvarious glycolytic metabolites proximal to the metabolic blockaccumulate in the red blood cells, while such cells become depleted ofthe distal products in the pathway, such as lactate and ATP. The lack ofATP disturbs the cation gradient across the red cell membrane, causingthe loss of potassium and water, which causes cell dehydration,contraction, and crenation, and leads to premature destruction of thered blood cell. The survival of patients with severe PK deficiencydepends on a compensatory expression of the PK-M2 isozyme, widelydistributed in various tissues, including red blood cells, in which thePK-M2 is the R isozyme.

Accordingly, there is a desire for new activators of PK-M2.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compounds that are activators of the M2isoform of human pyruvate kinase. In addition, the present inventionprovides compositions comprising these compounds and methods of usingthese compound as therapeutic agents in the treatment or prevention ofcancer.

The invention provides a compound of the formula (I):

wherein R¹ and R² are aryl or heteroaryl, optionally substituted withone or more substituents selected from the group consisting of C₁-C₁₀alkyl, C₃-C₆ alkylene, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ haloalkyl,C₁-C₁₀ dihaloalkyl, C₁-C₁₀ trihaloalkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, C₆-C₁₀ aryl, heterocyclyl, heteroaryl, heteroaryloxide,alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴, OCOR⁴, SCOR⁴, SOR⁴, SO₂R⁴,SO₂NR⁴R⁵, NO₂, B(OH)₂, CN, and halogen, and

L is a linker comprising an amino group;

or a pharmaceutically acceptable salt thereof.

The invention provides a compound of the formula (II):

wherein:

R¹¹ is selected from the group consisting of H, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀aryl, OR¹⁷, SR¹⁷, SOR¹⁷, SO₂R¹⁷, NR¹⁷R¹⁸, NCOR¹⁷, SCOR¹⁷, COR¹⁷, OCOR¹⁷,B(OH)₂, NO₂, NHCOR¹⁷, CN, CHO, hydroxy C₁-C₁₀ alkyl, and halogen,

R¹² is selected from the group consisting of H, C₁-C₂ alkyl, C₃-C₁₀cycloalkyl, NCOR¹⁴, and SO₂R¹⁴,

R¹³ to R¹⁶ are selected from the group consisting of H, C₁-C₁₀ alkyl,halo C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl,C₃-C₁₀ cycloalkenyl, C₆-C₁₀ aryl, heteroaryl, OR¹⁷, SR¹⁷, NR¹⁷R¹⁸,NCOR¹⁷, OCOR¹⁷, SCOR¹⁷, SOR¹⁷, SO₂R¹⁷, SO₂NR¹⁷R¹⁸, CF₃, and halogen, and

R¹⁷ and R¹⁸ are independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, and C₆-C₁₀ aryl,

or a pharmaceutically acceptable salt thereof.

The invention also provides a pharmaceutical composition comprising acompound or salt of the invention and a pharmaceutically acceptablecarrier.

The invention further provides a method for treating cancer comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a compound of the invention to a mammal afflicted therewith.

The invention additionally provides a method for treating certain formsof anemia associated with downregulation of the R form of pyruvatekinase.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1A illustrates that compound 1 increased the affinity of PKM2 forPEP, in accordance with an embodiment of the invention.

FIG. 1B illustrates the reduced effect of compound 1 on ADP kinetics.

FIG. 2 illustrates the selectivity of compound 1 to PKM1, in accordancewith another embodiment of the invention.

FIG. 3 illustrates qHTS data and its classification scheme rankingcriteria followed in this application.

FIG. 4A illustrates that compound 66 increased the affinity of PKM2 forPEP, in accordance with another embodiment of the invention.

FIG. 4B illustrates the reduced effect of compound 66 on ADP kinetics.

FIG. 5 illustrates the selectivity of compound 66 to PKM2, in accordancewith an embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an embodiment, the invention provides a compound ofFormula I:

wherein R¹ and R² are aryl or heteroaryl, optionally substituted withone or more substituents selected from the group consisting of C₁-C₁₀alkyl, C₃-C₁₀ alkylene, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀haloalkyl, C₁-C₁₀ dihaloalkyl, trihaloalkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, C₆-C₁₀ aryl, heterocyclyl, heteroaryl, heteroaryloxide,alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴, OCOR⁴, SCOR⁴, SOR⁴, SO₂R⁴,SO₂NR⁴R⁵, NO₂, B(OH)₂, CN, and halogen, and

L is a linker comprising an amino group;

or a pharmaceutically acceptable salt thereof

with the provisos that R¹ and R² are not dimethoxyphenyl or R¹ and R²are not both 4-methylphenyl simultaneously.

In accordance with an embodiment, L is a linear amino group, cyclicamino group, or a combination thereof.

In a particular embodiment, the compound of formula I is a compound offormula (Ia):

wherein n=1 to 3, R¹ and R² are aryl or heteroaryl optionallysubstituted with one or more substituents selected from the groupconsisting of C₁-C₁₀ alkyl, C₃-C₆ alkylene, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₁-C₁₀ haloalkyl, C₁-C₁₀ dihaloalkyl, C₁-C₁₀ trihaloalkyl,C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀ aryl, heterocyclyl,heteroaryl, heteroaryloxide, alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴,OCOR⁴, SCOR⁴, SOR⁴, SO₂R⁴, SO₂NR⁴R⁵, NO₂, B(OH)₂, CN, and halogen,

R³ and R⁴ are independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, COR⁶, F, and CF₃, or, R³ and R⁴, taken together, form C═O,

R⁵ and R⁷ to R¹⁰ are independently H, C₁-C₁₀ alkyl, or F,

R⁶ is C₁-C₁₀ alkyl or C₃-C₁₀ cycloalkyl, or

each of R⁷ and R⁸ and of R⁹ and R¹⁰, together form C═O and

X is CH or N,

or a pharmaceutically acceptable salt thereof.

In a specific embodiment, the compound or salt according to the abovedescribed embodiments is a compound wherein R¹ and R² are phenylsubstituted with one or more substituents selected from the groupconsisting of C₁-C₁₀ alkyl, C₁-C₁₀ trihaloalkyl, heterocyclyl,heteroaryl, alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴, OCOR⁴, SCOR⁴, SOR⁴,SO₂R⁴, SO₂NR⁴R⁵, CN, and halogen,

R³ and R⁴ are independently selected from the group consisting of H,C₁-C₁₀ alkyl, and F, or, taken together, form C═O, and

R⁵ and R⁷ to R¹⁰ are independently H, C₁-C₁₀ alkyl, or F.

In any of the embodiments above, R¹ and R² are phenyl substituted withone or more substituents selected from the group consisting of C₁-C₁₀alkyl, C₁-C₁₀ trihaloalkyl, heterocyclyl, heteroaryl, alkylenedioxy, CN,and halogen, and R³ to R¹⁰ are H.

In a particular embodiment of the compounds described above, X is N.

In a preferred embodiment of the compounds described above, n is 1.

Specific examples of the compounds described above include those whereinR¹ is selected from the group consisting of phenyl, 4-methylphenyl,2-methylphenyl, 2-fluorophenyl, 4-chlorophenyl, 4-fluorophenyl,4,2-difluorophenyl, 2,6-difluorophenyl, 2,4,5-trifluorophenyl,4-chloro-2-fluorophenyl, 3-chloro-2-fluorophenyl,4-trifluoromethylphenyl, 3-trifluoromethylphenyl,2,6-difluoro-4-trifluoromethylphenyl, 2,6-difluoro-4-methoxyphenyl,2,5-difluoro-4-propylphenyl, 2,6-difluoro-3-hydroxyphenyl,2,4-difluorophenyl, 4-bromo-2-fluorophenyl,2,6-difluoro-3-hydroxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl,4-cyanophenyl, 2-nitrophenyl, 2-pyridyl, 2-pyridyl-1-oxide, 2-(boronicacid)phenyl, 3-(boronic acid)phenyl, and 4-(boronic acid)phenyl;preferably wherein R¹ is selected from the group consisting of2,6-difluoro-4-trifluoromethylphenyl, 2,6-difluorophenyl,2,6-difluoro-4-methoxyphenyl, 2,6-difluoro-3-hydroxyphenyl, and4-methoxyphenyl.

In accordance with an embodiment, specific examples of the compound offormula Ia is wherein R¹ is heterocyclyl or heteroaryl, selected fromthe group consisting of 2-pyridyl, 2-pyridyl-N-oxide, 3-pyridyl,3-pyridyl-N-oxide, 4-pyridyl, 4-pyridyl-N-oxide, 2-pyrimidinyl,2-pyrimidinyl-N-oxide, 4-pyrimidinyl-N-oxide, 5-pyrimidinyl,pyrimidinyl-N-oxide, 2-pyrazinyl, and 2-pyrazinyl-N-oxide.

In any of the embodiments above, R² is6-(2,3-dihydrobenzo[b][1,4]dioxinyl),7-(3,4-dihydro-2H-benzo[b][1,4]dioxepinyl), 5-benzo[d][1,4]dioxinyl,7-(4-methyl-3,4-dihydro-2H-pyrido[3,2-b][1,4-oxazinyl), 2-naphthalenyl,6-(2,2-dimethylchromanyl), 5-(1-methyl-1H-indolyl),6-(2-methylbenzo[d]thiazolyl), or 4-methoxyphenyl, preferably6-(2,3-dihydrobenzo[b][1,4]dioxinyl).

In keeping with the embodiments described above, specific examples ofcompounds include compounds of formula (Ia), wherein X is N, n=1, and R³to R¹³ is H, and R¹ and R² are as follows:

R¹ is 4-methoxyphenyl and R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ and R² are 6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ and R² are 4-methoxyphenyl;

R¹ is 4-cyanophenyl and R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 4-chlorophenyl and R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 4-fluorophenyl and R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 3-fluorophenyl and R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 2-fluorophenyl and R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 2,6-difluorophenyl and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 2,4,5-trifluorophenyl and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 2,6-difluoro-4-methoxyphenyl and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 2,5-difluoro-3-propylphenyl and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 2,6-difluoro-3-hydroxyphenyl and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 2,4-difluorophenyl and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is phenyl and R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 3-(trifluoromethylphenyl) and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 3-methoxyphenyl and R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 4-methoxyphenyl and R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 2-pyridyl and R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 2-pyridyl-1-oxide and R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl);

R¹ is 2,6-difluorophenyl and R² is 2,6-difluorophenyl;

R¹ is 2,6-difluorophenyl and R² is7-(3,4-dihydro-2H-benzo[b][1,4]dioxepinyl);

R¹ is 2,6-difluorophenyl and R² is 5-benzo[d][1,4]dioxinyl;

R¹ is 2,6-difluorophenyl and R² is7-(4-methyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazinyl);

R¹ is 2,6-difluorophenyl and R² is 2-naphthalenyl;

R¹ is 2,6-difluorophenyl and R² is 6-(2,2-dimethylchromanyl);

R¹ is 2,6-difluorophenyl and R² is 5-(1-methyl-1H-indolyl);

R¹ is 2,6-difluorophenyl and R² is 6-(2-methylbenzo[d]thiazolyl); or

R¹ is 2,6-difluorophenyl and R² is 6-(2,3-dihydrobenzo[b][1,4]dioxinyl).

In accordance with another embodiment of the compound of formula Ia, Xis CH.

In a preferred embodiment, n is 1. In any of these embodiments,preferably R³, R⁴, and R⁵ are H. Examples of such compounds includethose wherein R¹ is selected from the group consisting of4-methylphenyl, 2-methylphenyl, 2-fluorophenyl, 3-fluorophenyl,4,2-difluorophenyl, 2,6-difluorophenyl, 2,4,5-trifluorophenyl,2,6-difluoro-4-trifluoromethylphenyl, 4-chloro-2-fluoro,3-chloro-2-fluoro, 4-trifluoromethylphenyl, 4-bromo-2-fluorophenyl,4-methoxyphenyl, and 2-nitrophenyl, particularly wherein R¹ is selectedfrom the group consisting of 2,6-difluoro-4-trifluoromethylphenyl,2,6-difluorophenyl, and 4-methoxyphenyl. In an embodiment of thesecompounds, R² is 3,4-ethylenedioxyphenyl.

In another embodiment of the compound of formula Ia is the compound offormula (Ib):

In a further embodiment and The compound or salt of claim 12, whereinthe compound is of formula (Ic):

wherein R³ to R¹⁰ are H or methyl, R³ to R⁶ and R⁹ and R¹⁰ are H ormethyl and R⁷ form C═O, or R³ to R⁸ are H or methyl and R⁹ and R¹⁰ formC═O.

In accordance with an embodiment of the compound of formula (Ic), (i) R⁵is methyl and R³, R⁴, and R⁶ to R¹⁰ are H; (ii) R⁶ is methyl and R³ toR⁵ and R⁷ to R¹⁰ are H; (iii) R³ is methyl and R⁴ to R¹⁰ are H; (iv) R⁴is methyl and R³ and R⁵ to R¹⁰ are H; (v) R³ to R⁸ are H and R⁹ and R¹⁰form C═O; or (vi) R³ to R⁶ and R⁷ and R⁸ are H and R⁷ and R⁸ form C═O.

In accordance with an embodiment of the compound of formula I, L is analkylene diamino group, cycloalkylamino amino, or cycloalkylaminoalkylamino. Examples of compounds of this embodiment include compoundswherein L is N,N′-(ethane-1,2-diyl), N,N′-(propane-1,3-diyl),N,N′-(butane-1,4-diyl), N,N′-(pentane-1,5-diyl), N,N′-(hexane-1,6-diyl),N,N′-((trans)-cyclohexane-1,4-diyl), N,N′-((cis)-cyclohexane-1,4-diyl),

In a specific embodiment of the above compounds, R¹ is2,6-difluorophenyl and R² is 6-(2,3-dihydrobenzo[b][1,4]dioxinyl)

In accordance with another embodiment, the invention provides a compoundof Formula II:

wherein:

R¹¹ is selected from the group consisting of H, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀aryl, OR¹⁷, SR¹⁷, SOR¹⁷, SO₂R¹⁷, NR¹⁷R¹⁸, NCOR¹⁷, SCOR¹⁷, COR¹⁷, OCOR¹⁷,B(OH)₂, NO₂, NHCOR¹⁷, CN, CHO, hydroxy C₁-C₁₀ alkyl, and halogen,

R¹² is selected from the group consisting of H, C₁-C₂ alkyl, C₃-C₁₀cycloalkyl, NCOR¹⁴, and SO₂R¹⁴,

R¹³ to R¹⁶ are selected from the group consisting of H, C₁-C₁₀ alkyl,halo C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl,C₃-C₁₀ cycloalkenyl, C₆-C₁₀ aryl, heteroaryl, OR¹⁷, SR¹⁷, NR¹⁷R¹⁸,NCOR¹⁷, OCOR¹⁷, SCOR¹⁷, SOR¹⁷, SO₂R¹⁷, SO₂NR¹⁷R¹⁸, CF₃, and halogen, and

R¹⁷ and R¹⁸ are independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, and C₆-C₁₀ aryl,

or a pharmaceutically acceptable salt thereof,

with the proviso that when R¹¹ is methyl, R¹² is methyl or allyl, andR¹⁴ to R¹⁶ are H, then R¹³ is not methoxy or fluoro.

In accordance with an embodiment of the compound of formula II, R¹¹ isselected from the group consisting of H, C₁-C₁₀ alkyl, OR¹⁷, SR¹⁷,SOR¹⁷, SO₂R¹⁷, NR¹⁷R¹⁸, NCOR¹⁷, SCOR¹⁷, COR¹⁷, OCOR¹⁷, B(OH)₂, NO₂,NHCOR¹⁷, CN, CHO, hydroxy C₁-C₁₀ alkyl, and halogen,

R¹² is selected from the group consisting of H, methyl, NCOR¹⁴, andSO₂R¹⁴,

R¹³ to R¹⁶ are selected from the group consisting of H, C₁-C₁₀ alkyl,OR¹⁷, SR¹⁷, NR¹⁷R¹⁸, NCOR¹⁷, OCOR¹⁷, SCOR¹⁷, SOR¹⁷, SO₂R¹⁷, SO₂NR¹⁷R¹⁸,CF₃, and halogen, and

R¹⁷ and R¹⁸ are independently selected from the group consisting of Hand C₁-C₁₀ alkyl.

In a particular embodiment of the compound of formula II, wherein R¹¹ isselected from the group consisting of H, C₁-C₁₀ alkyl, OR¹⁷, SR¹⁷,SOR¹⁷, COR¹⁷, OCOR¹⁷, B(OH)₂, NO₂, NHCOR¹⁷, CN, CHO, hydroxy C₁-C₁₀alkyl, and halogen, R¹² is H or C₁-C₂ alkyl, and

R¹³ to R¹⁶ are selected from the group consisting of H, methyl, CF₃,methoxy, and halogen.

Referring now to terminology used generically herein, for compounds offormula I or II, the term “alkyl” means a straight-chain or branchedalkyl substituent containing from, for example, 1 to about 6 carbonatoms, preferably from 1 to about 4 carbon atoms, more preferably from 1to 2 carbon atoms. Examples of such substituents include methyl, ethyl,propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl,isoamyl, hexyl, and the like.

The term “alkylene,” as used herein, means a cyclic alkylene group fusedto the phenyl group to which it is attached and containing from, forexample about 3 to about 5 carbon atoms, preferably from about 3 toabout carbon atoms. Examples of such substituents include, together withthe phenyl, dihydroindenyl and 1,2,3,4-tetrahydronaphthyl.

The term “alkenyl,” as used herein, means a linear alkenyl substituentcontaining at least one carbon-carbon double bond and from, for example,about 2 to about 6 carbon atoms (branched alkenyls are about 3 to about6 carbons atoms), preferably from about 2 to about 5 carbon atoms(branched alkenyls are preferably from about 3 to about 5 carbon atoms),more preferably from about 3 to about 4 carbon atoms. Examples of suchsubstituents include propenyl, isopropenyl, n-butenyl, sec-butenyl,isobutenyl, tert-butenyl, pentenyl, isopentenyl, hexenyl, and the like.

The term “alkynyl,” as used herein, means a linear alkynyl substituentcontaining at least one carbon-carbon triple bond and from, for example,2 to about 6 carbon atoms (branched alkynyls are about 3 to about 6carbons atoms), preferably from 2 to about 5 carbon atoms (branchedalkynyls are preferably from about 3 to about 5 carbon atoms), morepreferably from about 3 to about 4 carbon atoms. Examples of suchsubstituents include propynyl, isopropynyl, n-butynyl, sec-butynyl,isobutynyl, tert-butynyl, pentynyl, isopentynyl, hexynyl, and the like.

The term “cycloalkyl,” as used herein, means a cyclic alkyl substituentcontaining from, for example, about 3 to about 8 carbon atoms,preferably from about 4 to about 7 carbon atoms, and more preferablyfrom about 4 to about 6 carbon atoms. Examples of such substituentsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, and the like. The term “cycloalkenyl,” as used herein, meansthe same as the term “cycloalkyl,” however one or more double bonds arepresent. Examples of such substituents include cyclopentenyl andcyclohexenyl. The cyclic alkyl groups may be unsubstituted or furthersubstituted with alkyl groups such as methyl groups, ethyl groups, andthe like.

The term “heteroaryl,” as used herein, refers to a monocyclic orbicyclic 5- or 6-membered aromatic ring system containing one or moreheteroatoms selected from the group consisting of O, N, S, andcombinations thereof. Examples of suitable monocyclic heteroaryl groupsinclude but are not limited to furanyl, thiopheneyl, pyrrolyl,pyrazolyl, imidazolyl, 1,2,3-triazolyl, isoxazolyl, oxazolyl,isothiazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, andtriazinyl. The heteroaryl group can be attached to the sulfonamide groupat any available position on the heteroaryl group. For example, athiopheneyl group can be attached at the 2-position or the 3-position ofthe thiopheneyl group. A pyridyl group can be attached at the 2-, 3-, or4-position of the pyridyl group. Suitable bicyclic heterocycloarylgroups include monocyclic heterocycloaryl rings fused to a C₆-C₁₀ arylring. Non-limiting examples of bicyclic heterocycloaryl groups includebenzofuran, benzothiophene, quinoline, and isoquinoline. The heteroarylgroup is optionally substituted with 1, 2, 3, 4, or 5 substituents asrecited herein, wherein the optional substituent can be present at anyopen position on the heteroaryl group.

The term “heteroaryl oxide,” as used herein, refers to an oxidizedheteroaryl group as that term is defined herein, wherein one or more ofthe heteroatoms comprising the heteroaryl group is oxidized.Non-limiting examples of heteroaryl oxide groups include pyridineN-oxide, pyrimidine N-oxide, and pyrazine N-oxide.

The term “heterocyclyl” refers to a cyclic group, which may be aromaticor non-aromatic, or saturated or unsaturated, having one or moreheteroatoms such as O, N, or S. Examples of heterocyclyl groups includepyridyl, piperidinyl, piperazinyl, pyrazinyl, pyrolyl, pyranyl,tetrahydropyranyl, tetrahydrothiopyranyl, pyrrolidinyl, furanyl,tetrahydrofuranyl, thiophenyl, tetrahydrothiophenyl, purinyl,pyrimidinyl, thiazolyl, thiazolidinyl, thiazolinyl, oxazolyl, triazolyl,tetrazolyl, tetrazinyl, benzoxazolyl, morpholinyl, thiophorpholinyl,quinolinyl, and isoquinolinyl.

The term “halo” or “halogen,” as used herein, means a substituentselected from Group VIIA, such as, for example, fluorine, bromine,chlorine, and iodine.

The term “aryl” refers to an unsubstituted or substituted aromaticcarbocyclic substituent, as commonly understood in the art, and the term“C₆-C₁₀ aryl” includes phenyl and naphthyl. It is understood that theterm aryl applies to cyclic substituents that are planar and comprise4n+2π electrons, according to Mickel's Rule.

In a particular embodiment of the compound of formula II, R¹¹ isselected from the group consisting of H, methyl, ethyl, isopropyl, OCH₃,SCH₃, S(O)CH₃, NO₂, NHCOCH₃, CN, COOCH₃, CHO, CH₂OH, B(OH)₂, andCH(OH)CH₃, R¹² is methyl, R¹³ is 2-fluoro or chloro, and R¹⁴ to R¹⁶ areH.

In any of the embodiments of the compound of formula II, R¹¹ and R¹² aremethyl, R¹³ is H, 2-fluoro, 3-fluoro, 4-fluoro, 2-chloro, 3-chloro,4-chloro, 4-CF3, 4-methyl, or 4-methoxy, and R¹⁴ to R¹⁶ are H. Examplesof R¹ and R¹² are methyl, and of R¹³ and R¹⁴ are 2-fluoro and 4-fluoro,2-fluoro and 6-fluoro, 2-fluoro and 3-fluoro, 2-chloro and 6-fluoro,2-fluoro and 3-methyl, 2-fluoro and 4-methyl, 2-fluoro and 4-CF₃, and2-fluoro and 4-methoxy, and of R¹⁵ and R¹⁶ are H.

In a specific embodiment of the compound of the formula II, R¹¹ and R¹²are methyl, R¹³ to R¹⁵ are 2-fluoro, 3-fluoro, and 4-fluoro, and R¹⁶ isH. In another specific embodiment of the compound of formula II, R¹¹ andR¹² are methyl, R¹³ to R¹⁶ are 2-fluoro, 3-fluoro, 5-fluoro, and6-fluoro.

The present invention also provides a pharmaceutical compositioncomprising a compound or salt of any of the embodiments described aboveand a pharmaceutically acceptable carrier.

The present invention further provides a method of treating a diseaseresponsive to activation of human PK-M2 comprising administering to apatient in need thereof a therapeutically effective amount of a compoundof Formula I:

wherein R¹ and R² are aryl or heteroaryl, optionally substituted withone or more substituents selected from the group consisting of C₁-C₁₀alkyl, C₃-C₆ alkylene, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ haloalkyl,C₁-C₁₀ dihaloalkyl, C₁-C₁₀ trihaloalkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, C₆-C₁₀ aryl, heterocyclyl, heteroaryl, heteroaryloxide,alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴, OCOR⁴, SCOR⁴, SOR⁴, SO₂R⁴,SO₂NR⁴R⁵, NO₂, B(OH)₂, CN, and halogen, and

L is a linker comprising an amino group;

or a pharmaceutically acceptable salt thereof.

In accordance with an embodiment of the method, the compound is offormula Ia

wherein n=1 to 3, R¹ and R² are aryl, phenyl or heteroaryl, optionallysubstituted with one or more substituents selected from the groupconsisting of C₁-C₁₀ alkyl, C₃-C₆ alkylene, alkenyl, C₂-C₁₀ alkynyl,haloalkyl, C₁-C₁₀ dihaloalkyl, C₁-C₁₀ trihaloalkyl, C₃-C₁₀ cycloalkyl,C₃-C₁₀ cycloalkenyl, C₆-C₁₀ aryl, heterocyclyl, heteroaryl,heteroaryloxide, alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴, OCOR⁴, SCOR⁴,SOR⁴, SO₂R⁴, SO₂NR⁴R⁵, NO₂, B(OH)₂, CN, and halogen,

R³ and R⁴ are independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, COR⁶, F, and CF₃, or, R³ and R⁴, taken together, form C═O,

R⁵ and R⁷ to R¹⁰ are independently H, C₁-C₁₀ alkyl, or F,

R⁶ is C₁-C₁₀ alkyl or C₃-C₁₀ cycloalkyl, or

each of R⁷ and R⁸ and of R⁹ and R¹⁰, together form C═O

and

X is CH or N.

The present invention further provides a method of treating a diseaseresponsive to activation of human PK-M2 comprising administering to apatient in need thereof a therapeutically effective amount of a compoundof Formula II:

wherein:

R¹¹ is selected from the group consisting of H, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀aryl, OR¹⁷, SR¹⁷, SOR¹⁷, SO₂R¹⁷, NR¹⁷R¹⁸, NCOR¹⁷, SCOR¹⁷, COR¹⁷, OCOR¹⁷,B(OH)₂, NO₂, NHCOR¹⁷, CN, CHO, hydroxy C₁-C₁₀ alkyl, and halogen,

R¹² is selected from the group consisting of H, C₁-C₂ alkyl, C₃-C₁₀cycloalkyl, NCOR¹⁴, and SO₂R¹⁴,

R¹³ to R¹⁶ are selected from the group consisting of H, C₁-C₁₀ alkyl,halo C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl,C₃-C₁₀ cycloalkenyl, C₆-C₁₀ aryl, heteroaryl, OR¹⁷, SR¹⁷, NR¹⁷R¹⁸,NCOR¹⁷, OCOR¹⁷, SCOR¹⁷, SOR¹⁷, SO₂R¹⁷, SO₂NR¹⁷R¹⁸, CF₃, and halogen, and

R¹⁷ and R¹⁸ are independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, and C₆-C₁₀ aryl,

or a pharmaceutically acceptable salt thereof.

The invention further provides the use of a compound or apharmaceutically acceptable salt thereof in the manufacture of amedicament for treating a disease responsive to activation of humanPK-M2 of a patient, wherein the compound is of formula I:

wherein R¹ and R² are aryl or heteroaryl, optionally substituted withone or more substituents selected from the group consisting of C₁-C₁₀alkyl, C₃-C₆ alkylene, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, haloalkyl, C₁-C₁₀dihaloalkyl, C₁-C₁₀ trihaloalkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, C₆-C₁₀ aryl, heterocyclyl, heteroaryl, heteroaryloxide,alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴, OCOR⁴, SCOR⁴, SOR⁴, SO₂R⁴,SO₂NR⁴R⁵, NO₂, B(OH)₂, CN, and halogen, and

L is a linker comprising an amino group;

a compound of formula Ia:

wherein n=1 to 3, R¹ and R² are aryl, phenyl or heteroaryl, optionallysubstituted with one or more substituents selected from the groupconsisting of C₁-C₁₀ alkyl, C₃-C₆ alkylene, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₁-C₁₀ haloalkyl, C₁-C₁₀ dihaloalkyl, C₁-C₁₀ trihaloalkyl,C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀ aryl, heterocyclyl,heteroaryl, heteroaryloxide, alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴,OCOR⁴, SCO⁴, SOR⁴, SO₂R⁴, SO₂NR⁴R⁵, NO₂, B(OH)₂, CN, and halogen,

R³ and R⁴ are independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, COR⁶, F, and CF₃, or, R³ and R⁴, taken together, form C═O,

R⁵ and R⁷ to R¹⁰ are independently H, C₁-C₁₀ alkyl, or F,

R⁶ is C₁-C₁₀ alkyl or C₃-C₁₀ cycloalkyl, or

each of R⁷ and R⁸ and of R⁹ and R¹⁰, together form C═O

and

X is CH or N; or

a compound of formula II:

wherein:

R¹¹ is selected from the group consisting of H, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀aryl, OR¹⁷, SR¹⁷, SOR¹⁷, SO₂R¹⁷, NR¹⁷R¹⁸, NCOR¹⁷, SCOR¹⁷, COR¹⁷, OCOR¹⁷,B(OH)₂, NO₂, NHCOR¹⁷, CN, CHO, hydroxy C₁-C₁₀ alkyl, and halogen,

R¹² is selected from the group consisting of H, C₁-C₂ alkyl, C₃-C₁₀cycloalkyl, NCOR¹⁴, and SO₂R¹⁴,

R¹³ to R¹⁶ are selected from the group consisting of H, C₁-C₁₀ alkyl,halo C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl,C₃-C₁₀ cycloalkenyl, C₆-C₁₀ aryl, heteroaryl, OR¹⁷, SR¹⁷, NR¹⁷R¹⁸,NCOR¹⁷, OCOR¹⁷, SCOR¹⁷, SOR¹⁷, SO₂R¹⁷, SO₂NR¹⁷R¹⁸, CF₃, and halogen, and

R¹⁷ and R¹⁸ are independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, and C₆-C₁₀ aryl.

In accordance with a further embodiment, the invention provides acompound represented by Formula Id:

wherein n=1 to 3, R¹ and R² are phenyl substituted with one or moresubstituents selected from the group consisting of C₁-C₁₀ alkyl, C₃-C₆alkylene, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ haloalkyl, C₁-C₁₀dihaloalkyl, C₁-C₁₀ trihaloalkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, C₆-C₁₀ aryl, heteroaryl, heteroaryloxide, alkylenedioxy,OR⁴, SR⁴, NR⁴R⁵, NCOR⁴, OCOR⁴, SCOR⁴, SOR⁴, SO₂R⁴, SO₂NR⁴R⁵, nitro,boronic acid, and halogen,

R³ and R⁴ are independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, COR⁶, F, and CF₃, or, taken together, form C═O,

R⁵ is H, C₁-C₁₀ alkyl, or F,

R⁶ is C₁-C₁₀ alkyl or C₃-C₁₀ cycloalkyl, and

X is CH or N,

or a pharmaceutically acceptable salt thereof,

with the provisos that (1) when X is N, n=1, and R³, R⁴, and R⁵ are H orwhen X is N, n=1, and one of R³, R⁴, and R⁵ is alkyl, R¹ is notdimethoxyphenyl and (2) that R¹ and R² are not both 4-methylphenyl.

In certain embodiments of formula (Id), R¹ and R² are phenyl substitutedwith one or more substituents selected from the group consisting ofC₁-C₁₀ alkyl, C₁-C₁₀ trihaloalkyl, alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵,NCOR⁴, OCOR⁴, SCOR⁴, SOR⁴, SO₂R⁴, SO₂NR⁴R⁵, and halogen, wherein R³ andR⁴ are independently selected from the group consisting of H, C₁-C₁₀alkyl, and F, or, taken together, form C═O, and R⁵ is H, C₁-C₁₀ alkyl,or F.

In any of the embodiments of formula (Id), R¹ and R² are phenylsubstituted with one or more substituents selected from the groupconsisting of C₁-C₁₀ alkyl, C₁-C₁₀ trihaloalkyl, alkylenedioxy, andhalogen, and R³, R⁴, and R⁵ are H.

In certain embodiments of formula (Id), X is N and n is 1-3. Inaccordance with a preferred embodiment, n is 1. In a preferredembodiment, R¹ is selected from the group consisting of 4-methylphenyl,2-methylphenyl, 2-fluorophenyl, 3-fluorophenyl, 4,2-difluorophenyl,2,6-difluorophenyl, 2,4,5-trifluorophenyl, 4-chloro-2-fluorophenyl,3-chloro-2-fluorophenyl, 4-trifluoromethylphenyl,2,6-difluoro-4-trifluoromethylphenyl, 4-bromo-2-fluorophenyl,4-methoxyphenyl, 2-nitrophenyl, 2-(boronic acid)phenyl, 3-(boronicacid)phenyl, and 4-(boronic acid)phenyl. More preferably, R¹ is selectedfrom the group consisting of 2,6-difluoro-4-trifluoromethylphenyl,2,6-difluorophenyl, and 4-methoxyphenyl.

In a preferred embodiment of formula (Id), R² is3,4-ethylenedioxyphenyl.

In certain preferred embodiments of the compounds of formula (Id), theinvention provides a compound selected from the group consisting of1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(4-methylphenylphenylsulfonyl)piperazine,1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(2-methylphenylsulfonyl)piperazine,dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(2-fluorophenylsulfonyl)piperazine,1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(3-fluorophenylsulfonyl)piperazine,1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(2,4-difluorophenylsulfonyl)piperazine,1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(2,6-difluorophenylsulfonyl)piperazine,1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(2,4,5-trifluorophenylsulfonyl)piperazine,1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(4-chloro-2-fluorophenylsulfonyl)piperazine,1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(3-chloro-2-fluorophenylsulfonyl)piperazine,1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(4-trifluoromethylphenylsulfonyl)piperazine,1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(2,6-difluoro-4-trifluoromethylphenylsulfonyl)piperazine,1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(4-bromo-2-fluorophenylsulfonyl)piperazine,1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(4-methoxyphenylsulfonyl)piperazine,1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(2-nitrophenylsulfonyl)piperazine,1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(2-(boronicacid)phenylsulfonyl)piperazine,1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(3-(boronicacid)phenylsulfonyl)piperazine, and1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(4-(boronicacid)phenylsulfonyl)piperazine.

It will be understood that the terms 2-(boronic acid)phenyl, 3-(boronicacid)phenyl, and 4-(boronic acid)phenyl refer to a group of the formula:

wherein the phenyl group is attached to the sulfonyl group at the 2-,3-, or 4-position of the phenyl ring.

In certain embodiments of formula (Id), one of R³, R⁴, or R⁵ is C₁-C₁₀alkyl and two of R³, R⁴, and R⁵ are H. In certain preferred embodiments,the invention provides a compound selected from the group consisting of1-(2,6-difluorophenylsulfonyl)-4-(2,3-dihydrobenzo[b]dioxin-6-ylsulfonyl)-2-methylpiperazineor1-(2,6-difluorophenylsulfonyl)-4-(2,3-dihydrobenzo[b]dioxin-6-ylsulfonyl)-3-methylpiperazine.It will be recognized that when one of R³, R⁴, or R⁵ is C₁-C₁₀ alkyl,the carbon to which R³, R⁴, or R⁵ is C₁-C₁₀ alkyl is attached is achiral carbon center.

The invention contemplates embodiments in which a compound having achiral center is a substantially pure enantiomer thereof, a racemicmixture thereof, or a mixture containing any proportion of the twoenantiomers thereof.

In certain embodiments of formula (Id), one of R³, R⁴, or R⁵ is F. Inaccordance with these embodiments, two of R³, R⁴, or R⁵ areindependently H or C₁-C₁₀ alkyl, or when R⁵ is F, R³ and R⁴, takentogether, can be C═O.

In certain embodiments of formula (Id), R³ and R⁴, taken together, canbe C═O. In these embodiments, R⁵ is H, F, or C₁-C₁₀ alkyl. In a specificembodiment, the invention provides a compound that is1-(2,6-difluorophenylsulfonyl)-4-(2,3-dihydrobenzo[b]dioxin-6-ylsulfonyl)-3-oxopiperazine.

In certain embodiments of formula (Id), R¹ is selected from the groupconsisting of 2-pyridyl, 2-pyridyl-N-oxide, 3-pyridyl,3-pyridyl-N-oxide, 4-pyridyl, 4-pyridyl-N-oxide, 2-pyrimidinyl,2-pyrimidinyl-N-oxide, 4-pyrimidinyl-N-oxide, 5-pyrimidinyl,5-pyrimidinyl-N-oxide, 2-pyrazinyl, and 2-pyrazinyl-N-oxide. In apreferred embodiment, R¹ is selected from the group consisting of2-pyridyl, 3-pyridyl, and 4-pyridyl. In a more preferred embodiment, R¹is selected from the group consisting of 2-pyridyl-N-oxide,3-pyridyl-N-oxide, and 4-pyridyl-N-oxide. In these embodiments,preferably R² is 3,4-ethylenedioxyphenyl.

In certain embodiments of formula (Id), X is CH and n is 1-3. Inaccordance with a preferred embodiment, n is 1. In these embodiments,R¹, R², R³, R⁴, and R⁵ are as defined previously herein. In a specificembodiment, the invention provides a compound that is1-(2,6-difluorophenylsulfonyl)-4-(2,3-dihydrobenzo[b]dioxin-6-ylsulfonyl)-piperidine.

In accordance with another embodiment, the invention provides a compoundrepresented by Formula IIa:

wherein:

R⁷ is selected from the group consisting of H, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀aryl, OR¹⁰, SR¹⁰, SOR¹⁰, SO₂R¹⁰, NR¹⁰R¹¹, NCOR¹⁰, SCOR¹⁰, OCOR¹⁰,B(OH)₂, and halogen,

R⁸ is selected from the group consisting of C₁-C₁₀ alkyl, C₃-C₁₀cycloalkyl, NCOR¹⁰, and SO₂R¹⁰,

R⁹ is selected from the group consisting of C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀aryl, heteroaryl, OR¹⁰, SR¹⁰, NR¹⁰R¹¹, NCOR¹⁰, OCOR¹⁰, SCOR¹⁰, SOR¹⁰,SO₂R¹⁰, SO₂NR¹⁰R¹¹, CF₃, and halogen, and

R¹⁰ and R¹¹ are independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, and C₆-C₁₀ aryl,

or a pharmaceutically acceptable salt thereof,

with the proviso that when R⁷ is methyl and R⁸ is methyl or allyl, R⁹ isnot methoxy or fluoro.

In certain embodiments of formula (IIa), R⁷ is selected from the groupconsisting of H, C₁-C₁₀ alkyl, OR¹⁰, SR¹⁰, SOR¹⁰, SO₂R¹⁰, NR¹⁰R¹¹,NCOR¹⁰, SCOR¹⁰, OCOR¹⁰, B(OH)₂, and halogen, R⁸ is selected from thegroup consisting of C₁-C₁₀ alkyl, NCOR¹⁰, and SO₂R¹⁰, R⁹ is selectedfrom the group consisting of C₁-C₁₀ alkyl, OR¹⁰, SR¹⁰, NR¹⁰R¹¹, NCOR¹⁰,OCOR¹⁰, SCOR¹⁰, SOR¹⁰, SO₂R¹⁰, SO₂NR¹⁰R¹¹, CF₃, and halogen, and R¹⁰ andR¹¹ are independently selected from the group consisting of H and C₁-C₁₀alkyl. In preferred embodiments, R⁷ is selected from the groupconsisting of H, C₁-C₁₀ alkyl, or halogen, R⁸ is C₁-C₁₀ alkyl, and R⁹ isselected from the group consisting of C₁-C₁₀ alkyl, CF₃, and halogen.

In certain embodiments of formula (IIa), R⁹ is 2-fluoro. In accordancewith these embodiments, R⁷ is selected from the group consisting of H,Br, ethenyl, ethyl, propenyl, and propyl, and R⁸ is methyl. In specificembodiments, the invention provides a compound selected from the groupconsisting of4-methyl-4H-thieno[3,2-b]pyrrole-2-(2-fluorobenzyl)pyridazin-3(2H)one,2-bromo-4-methyl-4H-thieno[3,2-b]pyrrole-2-(2-fluorobenzyl)pyridazin-3(2H)one,4-methyl-2-vinyl-4H-thieno[3,2-b]pyrrole-2-(2-fluorobenzyl)pyridazin-3(2H)one,2-ethyl-4-methyl-4H-thieno[3,2-b]pyrrole-2-(2-fluorobenzyl)pyridazin-3(2H)one,4-methyl-(2-(prop-1-en-2-yl)-4H-thieno[3,2-b]pyrrole-2-(2-fluorobenzyl)pyridazin-3(2H)one,and2-isopropyl-4-methyl-4H-thieno[3,2-b]pyrrole-2-(2-fluorobenzyl)pyridazin-3(2H)one.

The present invention further provides a compound or a pharmaceuticallyacceptable salt thereof in the manufacture of a medicament for treatinga disease responsive to activation of human PK-M2 of a patient, whereinthe compound is of formula I:

wherein R¹ and R² are aryl or heteroaryl, optionally substituted withone or more substituents selected from the group consisting of C₁-C₁₀alkyl, C₃-C₆ alkylene, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ haloalkyl,C₁-C₁₀ dihaloalkyl, C₁-C₁₀ trihaloalkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, C₆-C₁₀ aryl, heterocyclyl, heteroaryl, heteroaryloxide,alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴, OCOR⁴, SCOR⁴, SOR⁴, SO₂R⁴,SO₂NR⁴R⁵, NO₂, B(OH)₂, CN, and halogen, and

L is a linker comprising an amino group;

a compound of formula Ia:

wherein n=1 to 3, R¹ and R² are aryl, phenyl or heteroaryl, optionallysubstituted with one or more substituents selected from the groupconsisting of C₁-C₁₀ alkyl, C₃-C₆ alkylene, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₁-C₁₀ haloalkyl, C₁-C₁₀ dihaloalkyl, C₁-C₁₀ trihaloalkyl,C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀ aryl, heterocyclyl,heteroaryl, heteroaryloxide, alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴,OCOR⁴, SCOR⁴, SOR⁴, SO₂R⁴, SO₂NR⁴R⁵, NO₂, B(OH)₂, CN, and halogen,

R³ and R⁴ are independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, COR⁶, F, and CF₃, or, R³ and R⁴, taken together, form C═O,

R⁵ and R⁷ to R¹⁰ are independently H, C₁-C₁₀ alkyl, or F,

R⁶ is C₁-C₁₀ alkyl or C₃-C₁₀ cycloalkyl, or

each of R⁷ and R⁸ and of R⁹ and R¹⁰, together form C═O

and

X is CH or N; or

a compound of formula II:

wherein:

R¹¹ is selected from the group consisting of H, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀aryl, OR¹⁷, SR¹⁷, SOR¹⁷, SO₂R¹⁷, NR¹⁷R¹⁸, NCOR¹⁷, SCOR¹⁷, COR¹⁷, OCOR¹⁷,B(OH)₂, NO₂, NHCOR¹⁷, CN, CHO, hydroxy C₁-C₁₀ alkyl, and halogen,

R¹² is selected from the group consisting of H, C₁-C₂ alkyl, C₃-C₁₀cycloalkyl, NCOR¹⁴, and SO₂R¹⁴,

R¹³ to R¹⁶ are selected from the group consisting of H, C₁-C₁₀ alkyl,halo C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl,C₃-C₁₀ cycloalkenyl, C₆-C₁₀ aryl, heteroaryl, OR¹⁷, SR¹⁷, NR¹⁷R¹⁸,NCOR¹⁷, OCOR¹⁷, SCOR¹⁷, SOR¹⁷, SO₂R¹⁷, SO₂NR¹⁷R¹⁸, CF₃, and halogen, and

R¹⁷ and R¹⁸ are independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, and C₆-C₁₀ aryl.

The present invention further provides a compound of formula III:

wherein R²¹ and R²² are aryl, substituted with one or more substituentsselected from the group consisting of C₁-C₁₀ alkyl, C₃-C₆ alkylene,C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ haloalkyl, C₁-C₁₀ dihaloalkyl,C₁-C₁₀ trihaloalkyl, C₃-C₁₀ cycloalkyl, cycloalkenyl, C₆-C₁₀ aryl,heterocyclyl, heteroaryl, heteroaryloxide, alkylenedioxy, OR²³, SR²³,NR²³R²⁴, NCOR²³, OCOR²³, SCOR²³, SO₂R²³, SO₂NR²³R²⁴, NO₂, B(OH)₂, CN andhalogen,

wherein R²³ and R²⁴ are independently H, C₁-C₁₀ alkyl, F, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, COR⁶,and CF₃,

or a pharmaceutically acceptable salt thereof.

In accordance with an embodiment of formula III, the invention providesthe following compound or salt thereof:

The present invention further provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a compound or saltas above described.

The present invention further provides a method of treating a diseaseresponsive to activation of human PKM2 comprising administering to apatient in need thereof a therapeutically effective amount of a compoundor salt as above described.

The present invention further provides for the use of a compound or saltas above described in the manufacture of a medicament for treating adisease responsive to activation of the human PKM2.

The phrase “pharmaceutically acceptable salt” is intended to includenontoxic salts synthesized from the parent compound which contains abasic or acidic moiety by conventional chemical methods. Generally, suchsalts can be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two.Generally, nonaqueous media such as ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 18th ed., Mack PublishingCompany, Easton, Pa., 1990, p. 1445, and Journal of PharmaceuticalScience, 66, 2-19 (1977).

Suitable bases include inorganic bases such as alkali and alkaline earthmetal bases, e.g., those containing metallic cations such as sodium,potassium, magnesium, calcium and the like. Non-limiting examples ofsuitable bases include sodium hydroxide, potassium hydroxide, sodiumcarbonate, and potassium carbonate. Suitable acids include inorganicacids such as hydrochloric acid, hydrobromic acid, hydroiodic acid,sulfuric acid, phosphoric acid, and the like, and organic acids such asp-toluenesulfonic, methanesulfonic acid, benzenesulfonic acid, oxalicacid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citricacid, benzoic acid, acetic acid, maleic acid, tartaric acid, fattyacids, long chain fatty acids, and the like. Preferred pharmaceuticallyacceptable salts of inventive compounds having an acidic moiety includesodium and potassium salts. Preferred pharmaceutically acceptable saltsof inventive compounds having a basic moiety (e.g., a pyridyl group)include hydrochloride and hydrobromide salts. The compounds of thepresent invention containing an acidic or basic moiety are useful in theform of the free base or acid or in the form of a pharmaceuticallyacceptable salt thereof.

It should be recognized that the particular counterion forming a part ofany salt of this invention is usually not of a critical nature, so longas the salt as a whole is pharmacologically acceptable and as long asthe counterion does not contribute undesired qualities to the salt as awhole.

It is further understood that the above compounds and salts may formsolvates, or exist in a substantially uncomplexed form, such as theanhydrous form. As used herein, the term “solvate” refers to a molecularcomplex wherein the solvent molecule, such as the crystallizing solvent,is incorporated into the crystal lattice. When the solvent incorporatedin the solvate is water, the molecular complex is called a hydrate.Pharmaceutically acceptable solvates include hydrates, alcoholates suchas methanolates and ethanolates, acetonitrilates and the like. Thesecompounds can also exist in polymorphic forms.

The present invention is further directed to a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atleast one compound or salt described herein.

It is preferred that the pharmaceutically acceptable carrier be one thatis chemically inert to the active compounds and one that has nodetrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularcompound of the present invention chosen, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of the pharmaceutical composition ofthe present invention. The following formulations for oral, aerosol,parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal,intrathecal, rectal, and vaginal administration are merely exemplary andare in no way limiting.

The pharmaceutical composition can be administered parenterally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration thatcomprise a solution of the inventive compound or salt dissolved orsuspended in an acceptable carrier suitable for parenteraladministration, including aqueous and non-aqueous isotonic sterileinjection solutions.

Overall, the requirements for effective pharmaceutical carriers forparenteral compositions are well known to those of ordinary skill in theart. See, e.g., Banker and Chalmers, eds., Pharmaceutics and PharmacyPractice, J. B. Lippincott Company, Philadelphia, pp. 238-250 (1982),and Toissel, ASHP Handbook on Injectable Drugs, 4th ed., pp. 622-630(1986). Such solutions can contain anti-oxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. The compound or salt of thepresent invention may be administered in a physiologically acceptablediluent in a pharmaceutical carrier, such as a sterile liquid or mixtureof liquids, including water, saline, aqueous dextrose and related sugarsolutions, an alcohol, such as ethanol, isopropanol, or hexadecylalcohol, glycols, such as propylene glycol or polyethylene glycol,dimethylsulfoxide, glycerol ketals, such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such aspoly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils useful in parenteral formulations include petroleum, animal,vegetable, or synthetic oils. Specific examples of oils useful in suchformulations include peanut, soybean, sesame, cottonseed, corn, olive,petrolatum, and mineral. Suitable fatty acids for use in parenteralformulations include oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acidesters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-beta-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations can contain preservatives and buffers. Inorder to minimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations will typically range fromabout 5 to about 15% by weight. Suitable surfactants includepolyethylene sorbitan fatty acid esters, such as sorbitan monooleate andthe high molecular weight adducts of ethylene oxide with a hydrophobicbase, formed by the condensation of propylene oxide with propyleneglycol. The parenteral formulations can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials, and can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid excipient, for example, water, forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions can be prepared from sterile powders, granules, andtablets of the kind previously described.

Topical formulations, including those that are useful for transdermaldrug release, are well-known to those of skill in the art and aresuitable in the context of the invention for application to skin.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as a therapeutically effective amount of the inventivecompound dissolved in diluents, such as water, saline, or orange juice,(b) capsules, sachets, tablets, lozenges, and troches, each containing apredetermined amount of the active ingredient, as solids or granules,(c) powders, (d) suspensions in an appropriate liquid, and (e) suitableemulsions. Liquid formulations may include diluents, such as water andalcohols, for example, ethanol, benzyl alcohol, and the polyethylenealcohols, either with or without the addition of a pharmaceuticallyacceptable surfactant, suspending agent, or emulsifying agent. Capsuleforms can be of the ordinary hard- or soft-shelled gelatin typecontaining, for example, surfactants, lubricants, and inert fillers,such as lactose, sucrose, calcium phosphate, and corn starch. Tabletforms can include one or more of lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,and other excipients, colorants, diluents, buffering agents,disintegrating agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible excipients. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to the activeingredient, such excipients as are known in the art.

The compound or salt of the present invention, alone or in combinationwith other suitable components, can be made into aerosol formulations tobe administered via inhalation.

The compounds are preferably supplied in finely divided form along witha surfactant and propellant. Typical percentages of active compound are0.01%-20% by weight, preferably 1%-10%. The surfactant must, of course,be nontoxic, and preferably soluble in the propellant. Representative ofsuch surfactants are the esters or partial esters of fatty acidscontaining from 6 to 22 carbon atoms, such as caproic, octanoic, lauric,palmitic, stearic, linoleic, linolenic, olesteric and oleic acids withan aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters,such as mixed or natural glycerides may be employed. The surfactant mayconstitute 0.1%-20% by weight of the composition, preferably 0.25%-5%.The balance of the composition is ordinarily propellant. A carrier canalso be included as desired, e.g., lecithin for intranasal delivery.These aerosol formulations can be placed into acceptable pressurizedpropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike. They also may be formulated as pharmaceuticals for non-pressuredpreparations, such as in a nebulizer or an atomizer. Such sprayformulations may be used to spray mucosa.

Additionally, the compound or salt of the present invention may be madeinto suppositories by mixing with a variety of bases, such asemulsifying bases or water-soluble bases. Formulations suitable forvaginal administration may be presented as pessaries, tampons, creams,gels, pastes, foams, or spray formulas containing, in addition to theactive ingredient, such carriers as are known in the art to beappropriate.

It will be appreciated by one of ordinary skill in the art that, inaddition to the aforedescribed pharmaceutical compositions, the compoundor salt of the present invention may be formulated as inclusioncomplexes, such as cyclodextrin inclusion complexes, or liposomes.Liposomes serve to target the compounds to a particular tissue, such aslymphoid tissue or cancerous hepatic cells. Liposomes can also be usedto increase the half-life of the inventive compound. Liposomes useful inthe present invention include emulsions, foams, micelles, insolublemonolayers, liquid crystals, phospholipid dispersions, lamellar layersand the like. In these preparations, the active agent to be delivered isincorporated as part of a liposome, alone or in conjunction with asuitable chemotherapeutic agent. Thus, liposomes filled with a desiredinventive compound or salt thereof, can be directed to the site of aspecific tissue type, hepatic cells, for example, where the liposomesthen deliver the selected compositions. Liposomes for use in theinvention are formed from standard vesicle-forming lipids, whichgenerally include neutral and negatively charged phospholipids and asterol, such as cholesterol. The selection of lipids is generally guidedby consideration of, for example, liposome size and stability of theliposomes in the blood stream. A variety of methods are available forpreparing liposomes, as described in, for example, Szoka et al., Ann.Rev. Biophys. Bioeng., 9, 467 (1980), and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369. For targeting to the cells of aparticular tissue type, a ligand to be incorporated into the liposomecan include, for example, antibodies or fragments thereof specific forcell surface determinants of the targeted tissue type. A liposomesuspension containing a compound or salt of the present invention may beadministered intravenously, locally, topically, etc. in a dose thatvaries according to the mode of administration, the agent beingdelivered, and the stage of disease being treated.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages that are lessthan the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstances is reached. The present inventive method typically willinvolve the administration of about 0.1 to about 300 mg of one or moreof the compounds described above per kg body weight of the individual.

The invention further provides a method for treating a diseaseresponsive to activation of PK-M2 in a mammal comprising administeringan effective amount of the compound of the invention to a mammalafflicted therewith. In accordance with an embodiment, the inventionprovides a method of treating a disease responsive to activation ofPK-M2 comprising administering to a patient in need thereof atherapeutically effective amount of a compound represented by FormulaIa:

wherein n=1 to 3, R¹ and R² are aryl, phenyl, or heteroaryl, substitutedwith one or more substituents selected from the group consisting ofC₁-C₁₀ alkyl, C₃-C₆ alkylene, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀haloalkyl, C₁-C₁₀ dihaloalkyl, C₁-C₁₀ trihaloalkyl, C₃-C₁₀ cycloalkyl,C₃-C₁₀ cycloalkenyl, C₆-C₁₀ aryl, heteroaryl, heteroaryloxide,alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴, OCOR⁴, SCOR⁴, SOR⁴, SO₂R⁴,SO₂NR⁴R⁵, nitro, boronic acid, and halogen,

R³ and R⁴ are independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, COR⁶, F, and CF₃, or, taken together, form C═O,

R⁵ is H, C₁-C₁₀ alkyl, or F,

R⁶ to R¹⁰ are H, and

X is CH or N,

or a pharmaceutically acceptable salt thereof.

In accordance with another embodiment, the invention provides a methodof treating a disease responsive to activation of PK-M2 comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a compound represented by Formula II:

wherein:

R¹¹ is selected from the group consisting of H, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀aryl, OR¹⁷, SR¹⁷, SOR¹⁷, SO₂R¹⁷, NR¹⁷R¹⁸, NCOR¹⁷, SCOR¹⁷, OCOR¹⁷,B(OH)₂, and halogen,

R¹² is selected from the group consisting of C₁-C₁₀ alkyl, C₃-C₁₀cycloalkyl, NCOR¹⁷, and SO₂R¹⁷,

R¹³ is selected from the group consisting of C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀aryl, heteroaryl, OR¹⁷, SR¹⁷, NR¹⁷R¹⁸, NCOR¹⁷, OCOR¹⁷, SCOR¹⁷, SOR¹⁷,SO₂R¹⁷, SO₂NR¹⁷R¹⁸, CF₃, and halogen, and

R¹⁷ and R¹⁸ are independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, and C₆-C₁₀ aryl,

or a pharmaceutically acceptable salt thereof.

The disease responsive to activation of PK-M2 can be cancer or anemia.The cancer can be any suitable cancer, for example, renal cancer,ovarian cancer, breast cancer, CNS cancer, leukemia, prostate cancer,non-small cell lung cancer, colon cancer, or melanoma, particularlyrenal cancer, CNS cancer, breast cancer, and ovarian cancer. The anemiacan be any suitable anemia, for example hemolytic anemia such as humanerythrocyte R-type pyruvate kinase deficiency.

The invention further provides a use of a compound or salt of theinvention in the manufacture of a medicament for treating diseaseresponsive to activation of PK-M2. The medicament typically is apharmaceutical composition as described herein.

One skilled in the art will appreciate that suitable methods ofutilizing a compound and administering it to a human for the treatmentof disease states, in particular, diseases responsive to activation ofPK-M2, which would be useful in the method of the present invention, areavailable. Although more than one route can be used to administer aparticular compound, a particular route can provide a more immediate andmore effective reaction than another route. Accordingly, the describedmethods are merely exemplary and are in no way limiting.

The dose administered to a human in accordance with the presentinvention should be sufficient to effect the desired response. Suchresponses include reversal or prevention of the bad effects of thedisease responsive to activation of PK-M2 for which treatment is desiredor to elicit the desired benefit. One skilled in the art will recognizethat dosage will depend upon a variety of factors, including the age,condition, and body weight of the human, as well as the source,particular type of the cancer, and extent of cancer in the human. Thesize of the dose will also be determined by the route, timing andfrequency of administration as well as the existence, nature, and extentof any adverse side-effects that might accompany the administration of aparticular compound and the desired physiological effect. It will beappreciated by one of skill in the art that various conditions ordisease states may require prolonged treatment involving multipleadministrations.

The compounds of the invention can prepared by any suitable method. Forexample, the N,N′-diarylsulfonamides were prepared by a sequence ofcoupling reaction, deprotection and a second coupling reaction asdetailed in Scheme 1. Specifically, mono-boc protected piperazine inmethylene chloride at 0° C. in the presence of triethylamine was coupledto numerous aryl sulfonyl chlorides to provide the needed boc-protectedN-arylsulfonamides. These intermediates were deprotected with TFA inmethylene chloride at 0° C. and subsequently coupled to a second arylsulfonyl chloride to provide the N,N′-diarylsulfonamide analogues. Allfinal compounds were purified by preparative scale HPLC and the yieldsfor these procedures were typically high. The same procedure wasutilized to explore alternate ligations between each aryl-sulfonamidemoiety including cyclic diamines of different ring size (analogue 31),linear diamines (analogue 32-36), ring systems with an internalsecondary amine and an exocyclic amine (analogues 37-44), and analogueswith variously substituted piperazines (analogues 45-47) (scheme notshown). Several of the related sulfone derivatives akin to the leadstructure were made according to Scheme 2. To synthesize thesederivatives N-boc-4-bromopiperidine was treated with various arylsulfides in basic DMF to afford the appropriately substituted thiolethers. Oxidation to the sulfone was accomplished by reaction with mCPBAin methylene chloride at 0° C. Following boc deprotection the secondaryamine was coupled to various aryl sulfonyl chlorides to provide the4-(arylsulfonyl)-1-(arylsulfonyl)piperidine analogues (represented byanalogues 20 and 30). N,N′-diarylsulfonamide analogues having thepiperazin-2-one core were prepared according to Scheme 3. Thesederivatives were accessed through treatment of piperazin-2-one with 1equivalent of various aryl sulfonyl chlorides which preferentiallycoupled to the free amine moiety. The resulting intermediate wasconverted to the 1,4-bis(arylsulfonyl)piperazin-2-ones by deprotonationof the amide with LHMDS in THF at −78° C. followed by addition ofvarious aryl sulfonyl chlorides to generate the desired products in goodyields (represented by analogues 49 and 50).

Compounds of formula II, i.e., thethieno[3,2-b]pyrrole[3,2-d]pyridazinones and analogues were prepared asfollows. The sequence required for the chemical synthesis ofNCGC00031955 66 was according to Scheme 4. Several commerciallyavailable thiophene-2-carbaldehydes were reacted with ethyl2-azidoacetate in sodium ethoxide at 0° C. to provide the corresponding2-azido-3-(thiophen-2-yl)acrylates. Refluxing this intermediate ino-xylene provided the core thienopyrroles in good yields.Vilsmeier-Haack reaction was used to form the substituted ethyl6-formyl-4H-thieno[3,2-b]pyrrole-5-carboxylates. There was no indicationof alternate regiochemical acyl insertion. Through a series ofexperiments, it was necessary to alkylate the pyrrole nitrogen beforeproceeding with the synthesis. This was accomplished via treatment withalkyl iodides in basic DMF. The remainder of the synthesis involved theformation of the pyridazinone via treatment with hydrazine in refluxing2-ethoxyethanol and alkylation of the amide nitrogen with various alkyland benzyl bromides in basic DMF. Arylation of the amide nitrogen wasalso explored through a copper catalyzed process developed by Buchwaldand coworkers (J. Am. Chem. Soc., 123, 7727-7729).

The utility of 5-bromothiophene-2-carbaldehyde as a starting reagent inthis sequence was a key to the synthetic elaboration of numerousanalogues (Scheme 5). From the 2-bromo final product we conductednumerous transformations. Treatment with sodium methoxide in refluxing1,4-dioxane in the presence of copper iodide provided the 2-methoxyderivative 71 in good yield. Copper catalysis was again used for theinsertion of acetamide to provide direct access to the NHAc derivative84. The nitrile analogue 82 was achieved through treatment of thebromide with CuCN in DMF at elevated temperatures. Palladium (0)catalysis, carbon monoxide and triethylamine in a MeOH/DMSO solutionproved to be a successful strategy to insert the methyl ester moiety of83. To obtain compounds having various substituents at the 5-position ofthe thiophene in the final product, either vinyl or isopropenylboronicacids pinacol esters were entered into traditional Suzuki-Miyauracouplings to provide derivatives that, upon reduction, yielded the ethylor isopropyl derivatives 68 and 69. Using the identical reductiveconditions of the starting bromide provided analogue 70 for study.Creation of the Grignard reagent was accomplished through metal-halogenexchange and exposure of this intermediate to trimethyl borate at 0° C.followed by work-up in 0.1 N aqueous HCl provided the boronic acidanalogue 86. Alternatively, quenching of the Grignard reagent withformaldehyde provided the secondary alcohol 88 which was furtheroxidized to the ketone 87 with IBX in DMSO. Treatment of the bromidewith sodium methanethiolate with copper(I) bromide in DMF at 140° C.provided the thiol ether 72 and mCPBA oxidation yielded the sulfoxide 73and sulfone 74 which were separable through chromatographic methods.

Nitration following insertion of the aldehyde moiety at the 6-positionprovided nitration to the appropriate 2-position of the heterocycle(Scheme 6). The formation of the pyridazinone ring was more facileproceeding in ethanol at room temperature.

To insert hydrogen bond donors into the core structure, theun-substituted derivative 71 was entered into a second Vilsmeier-Haackreaction at the 2-position of the thiophene ring to produce the aldehyde84 (Scheme 7). Reduction of this agent with sodium borohydride inmethanol provided the alcohol 88 for examination.

Changes were also made directly on or to the pyridazinone ring. The sixposition of this ring system was the only position open formodification. To examine if substituents could be added at the loneun-substituted carbon the aldehyde was converted to the methyl ketonethrough addition of a methyl Grignard reagent and IBX oxidation of theresulting secondary alcohol (Scheme 8). From this intermediate, steps ivthrough vi of Scheme 1 were used to produce the 6-methyl version of ourlead compound. A second consideration was changing from a pyridazinoneto a pyrimidinone ring system (Scheme 9). To accomplish this, we tookadvantage of our observation that nitration of the ethyl4H-thieno[3,2-b]pyrrole-5-carboxylate intermediate occurred on the 6position of the pyrrole ring. Reduction of the nitro group was achievedvia treatment with tin (II) chloride in acidic EtOH/H₂O and thepyrimidinone ring was formed upon condensation with ammonia formate andformamide at elevated temperatures. The benzylation of the amidenitrogen occurred under similar conditions.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Unless otherwise noted, all reactions were carried out under anatmosphere of dry argon or nitrogen in dried glassware. Indicatedreaction temperatures refer to those of the reaction bath, while roomtemperature (RT) is noted as 25° C. All solvents were of anhydrousquality purchased form Aldrich Chemical Co. and used as received.Commercially available starting materials and reagents were purchasedform Aldrich, TCI and Acros and were used as received.

Example 1

This example illustrates general methods in preparing compounds of theinvention in accordance with an embodiment.

All air or moisture sensitive reactions were performed under positivepressure of nitrogen with oven-dried glassware. Anhydrous solvents suchas tetrahydrofuran (THF), toluene, dichloromethane, N,N-dimethylforamide(DMF), acetonitrile, methanol and triethylamine were obtained bypurchasing from Sigma-Aldrich. Preparative purification was performed ona Waters semi-preparative HPLC. The column used was a Phenomenex LunaC18 (5 micron, 30×75 mm) at a flow rate of 45 mL/min. The mobile phaseconsisted of acetonitrile and water (each containing 0.1%trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8minutes was used during the purification. Fraction collection wastriggered by UV detection (220 nM). Analytical analysis was performed onan Agilent LC/MS (Agilent Technologies, Santa Clara, Calif.). Method 1:A 7 minute gradient of 4% to 100% Acetonitrile (containing 0.025%trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid)was used with an 8 minute run time at a flow rate of 1 mL/min. APhenomenex Luna C18 column (3 micron, 3×75 mm) was used at a temperatureof 50° C. Method 2: A 3 minute gradient of 4% to 100% Acetonitrile(containing 0.025% trifluoroacetic acid) in water (containing 0.05%trifluoroacetic acid) was used with a 4.5 minute run time at a flow rateof 1 mL/min. A Phenomenex Gemini Phenyl column (3 micron, 3×100 mm) wasused at a temperature of 50° C. Purity determination was performed usingan Agilent Diode Array Detector. Mass determination was performed usingan Agilent 6130 mass spectrometer with electrospray ionization in thepositive mode. ¹H NMR spectra were recorded on Varian 400 MHzspectrometers. Chemical Shifts are reported in ppm withtetramethylsilane (TMS) as internal standard (0 ppm) for CDCl₃ solutionsor undeuterated solvent (DMSO-h6 at 2.49 ppm) for DMSO-d6 solutions. Allof the analogs for assay have purity greater than 95% based on bothanalytical methods. High resolution mass spectrometry was recorded onAgilent 6210 Time-of-Flight LC/MS system. Confirmation of molecularformula was accomplished using electrospray ionization in the positivemode with the Agilent Masshunter software (version B.02).

Most bis-sulfonamides were synthesized by a three-step, two-potprocedure (Method A and Method B) exemplified by the synthesis of 1,shown in Schemes 1-3.

Method A:

1-Boc-piperazine (250 mg, 1.34 mmol, 1 equiv.) was dissolved indichloromethane (2.5 mL) and cooled in an ice bath under nitrogenatmosphere. Triethylamine (375 μl, 2.68 mmol, 2.0 equiv.) was addedfollowed by portionwise addition of2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride (346 mg, 1.48 mmol,1.1 equiv.). The reaction was stirred in the ice bath for one hour, thenquenched with saturated aqueous ammonium chloride (˜3 mL). The organiclayer was washed twice with saturated ammonium chloride, once withbrine, dried over sodium sulfate and concentrated in vacuo and thenpurified on silica gel chromatography using a 95/5-5/95, hexane/EtOAc(v/v) gradient to give1-boc-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazine as awhite powder (516 mg, 89% yield).

Method B:

1-Boc-4-(2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl)piperazine (400 mg,1.04 mmol) was dissolved in dichloromethane (1 mL) and cooled in an icebath. Trifluoroacetic acid (1 mL) was then added and the solution wasstirred in the ice bath. The reaction was monitored by TLC and showedcompletion after one hour. The solution was removed from the ice bathand the solvents removed on in vacuo to yield the TFA salt of1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazine, which wascarried onto the next step without purification. The oily residue wasdissolved in dichloromethane (2 mL) and cooled in an ice bath.Triethylamine (580 μl, 4.16 mmol, 4 equiv.) was added followed byportionwise addition of 4-methoxybenzene-1-sulfonyl chloride (236 mg,1.14 mmol, 1.1 equiv.). The progress was monitored by TLC and showedcompletion after 1 hour. The reaction was quenched with saturatedaqueous ammonium chloride (˜3 mL). The organic layer was washed twicewith saturated ammonium chloride, once with brine, dried over sodiumsulfate and concentrated in vacuo and then dissolved in DMSO andpurified by reverse phase HPLC.

Synthesis of Sulfone 30

4-bromo-1-boc piperidine (500 mg, 1.89 mmol, 1 equiv.) and2,3-dihydrobenzo[b][1,4]dioxine-6-thiol (318 mg, 1.89 mmol, 1 equiv.)were dissolved in DMF (4 mL). Potassium carbonate (392 mg, 2.84 mmol,1.5 equiv.) was then added and the solution was stirred at 80° C. for 5hours. The reaction was cooled to room temperature, diluted with ethylacetate (˜10 mL) and water (˜10 mL). The organic layer was washed withsaturated aqueous sodium bicarbonate, brine, dried over sodium sulfateand the solvents removed. The crude sulfide was dissolved indichloromethane (6 mL) and cooled to 0° C. Solid m-CPBA (720 mg, 4.16mmol, 2.2 equiv. based on initial thiol) was then added and thesuspension stirred at 0° C. for 2 hours. The suspension was thenfiltered and the filtrate was washed with 10% aqueous sodiumthiosulfate, aqueous sodium bicarbonate, brine and dried over sodiumsulfate. The solvent was removed and the residue was purified by silicagel chromatography using a 95/5-5/95, hexane/EtOAc (v/v) gradient togive the desired sulfone. Method B (see above) was then used to cleavethe boc-group and introduce the sulfonamide moiety (by using2,6-difluorobenzenesulfonyl chloride) to give product 30 which wasdissolved in DMSO and purified by reverse phase HPLC.

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(4-methoxyphenylsulfonyl)piperazine(1). ¹H NMR (400 MHz, DMSO-d₆) δ: 7.67-7.57 (m, 2H), 7.19-7.08 (m, 4H),7.08-7.01 (m, 1H), 4.45-4.23 (m, 4H), 3.86 (s, 3H), 2.94 (m, 8H). LC/MS:Method 1, retention time: 5.744 min; Method 2, retention time: 3.889min. HRMS: m/z (M+)=454.0872 (Calculated for C₁₉H₂₂N₂O₇S₂=454.0868).

1,4-bis(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazine (2). ¹HNMR (400 MHz, DMSO-d₆) δ: 7.70-7.61 (m, 2H), 7.61-7.52 (m, 1H),7.22-7.12 (m, 2H), 7.12-7.05 (m, 1H), 4.35 (m, 8H), 3.44-3.36 (m, 4H),3.00-2.88 (m, 4H). LC/MS: Method 1, retention time: 6.114 min; Method 2,retention time: 3.961 min. HRMS: m/z (M+)=482.0816 (Calculated forC₂₀H₂₂N₂O₈S₂ 482.0818).

1,4-bis(4-methoxyphenylsulfonyl)piperazine (3). ¹H NMR (400 MHz,DMSO-d₆) δ: 7.58 (d, 4H, J=6.9 Hz), 7.08 (d, 4H, J=8.4 Hz), 3.82 (s,6H), 2.91 (s, 8H). LC/MS: Method 1, retention time: 5.828 min; Method 2,retention time: 3.895 min.

4-(4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazin-1-ylsulfonyl)benzonitrile(4). ¹H NMR (400 MHz, DMSO-d₆) δ: 8.07 (d, 2H, J=8.4 Hz), 7.84 (d, 2H,J=8.4 Hz), 7.10 (m, 2H), 7.00 (m, 1H), 4.31 (m, 4H), 3.03-2.91 (m, 8H).LC/MS: Method 1, retention time: 5.671 min; Method 2, retention time:3.879 min. HRMS: m/z (M+)=449.0716 (Calculated forC₁₉H₁₉N₃O₆S₂=449.0715).

1-(4-chlorophenylsulfonyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazine(5). ¹H NMR (400 MHz, DMSO-d₆) δ: 7.67 (b, 4H), 7.12 (m, 2H), 7.03 (m,1H), 4.32 (m, 4H), 2.95 (m, 8H). LC/MS: Method 1, retention time: 6.114min; Method 2, retention time: 3.959 min. HRMS: m/z (M+)=458.0380(Calculated for C₁₈H₁₉ClN₂O₆S₂=458.0373).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(4-fluorophenylsulfonyl)piperazine(6). ¹H NMR (400 MHz, DMSO-d₆) δ: 7.72 (m, 2H), 7.43 (m, 2H), 7.18-7.09(m, 2H), 7.09-7.01 (m, 1H), 4.32 (m, 4H), 2.93 (m, 8H). LC/MS: Method 1,retention time: 5.813 min; Method 2, retention time: 3.893 min. HRMS:m/z (M+)=442.0677 (Calculated for C₁₈H₁₉FN₂O₆S₂=442.0669).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(3-fluorophenylsulfonyl)piperazine(7). ¹H NMR (400 MHz, DMSO-d₆) δ: 7.75-7.64 (m, 1H), 7.64-7.49 (m, 3H),7.18-7.09 (m, 2H), 7.09-7.01 (m, 1H), 4.43-4.25 (m, 4H), 3.12-3.00 (m,4H), 2.99-2.82 (m, 4H). LC/MS: Method 1, retention time: 5.853 min;Method 2, retention time: 3.911 min. HRMS; m/z (M+)=442.0662 (Calculatedfor C₁₈H₁₉FN₂O₆S₂=442.0669).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(2-fluorophenylsulfonyl)piperazine(8). ¹H NMR (400 MHz, DMSO-d₆) δ: 7.84-7.68 (m, 2H), 7.52-7.36 (m, 2H),7.21-7.11 (m, 2H), 7.10-7.02 (m, 1H), 4.44-4.25 (m, 4H), 3.23-3.07 (m,4H), 3.04-2.87 (m, 4H), LC/MS: Method 1, retention time: 5.775 min;Method 2, retention time: 3.891 min. HRMS; m/z (M+)=442.0664 (Calculatedfor C₁₈H₁₉FN₂O₆S₂=442.0669).

1-(2,6-difluorophenylsulfonyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazine(9). ¹H NMR (400 MHz, CDCl₃) δ: 7.55 (m, 1H), 7.24 (m, 2H), 7.00 (m,3H), 4.33 (m, 4H), 3.38 (m, 4H), 3.13 (m, 4H). LC/MS: Method 1,retention time: 5.781 min; Method 2, retention time: 3.889 min. HRMS:m/z (M+)=460.0570 (Calculated for C₁₈H₁₈F₂N₂O₆S₂=460.0574).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(2,4,5-trifluorophenyl)sulfonyl)piperazine(10). ¹H NMR (400 MHz, DMSO-d₆) δ: 8.00-7.76 (m, 2H), 7.22-7.12 (m, 2H),7.11-7.04 (m, 1H), 4.34 (dd, 4H, J=12.13, 5.09 Hz), 3.24-3.14 (m, 4H),3.04-2.87 (m, 4H). LC/MS: Method 1, retention time: 6.076 min; Method 2,retention time: 3.936 min. HRMS; m/z (M+)=478.0495 (Calculated forC₁₈H₁₇F₃N₂O₆S₂=478.0480).

1-(2,6-difluoro-4-methoxyphenylsulfonyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazine(11). ¹H NMR (400 MHz, CDCl₃) δ: 7.22 (m, 2H), 6.97 (m, 1H), 6.53 (d,2H, J=10.56 Hz), 4.26 (m, 4H), 3.87 (s, 3H), 3.31 (m, 4H), 3.11 (m, 4H).LC/MS: Method 1, retention time: 5.922 min; Method 2, retention time:3.911 min. HRMS; m/z (M+)=490.0698 (Calculated forC₁₉H₂₀F₂N₂O₇S₂=490.0680).

1-(2,5-difluoro-4-propylphenylsulfonyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazine(12). ¹H NMR (400 MHz, CDCl₃) δ: 7.44 (dd, 1H, J=8.41, 5.67 Hz), 7.23(m, 2H), 7.06 (dd, 1H, J=10.17, 5.48 Hz), 6.98 (d, 1H, J=8.22 Hz), 4.32(m, 4H), 3.30 (m, 4H), 3.11 (m, 4H), 2.66 (t, 2H, J=7.43 Hz), 1.66 (m,2H), 0.98 (t, 3H, J=7.43 Hz). LC/MS: Method 1, retention time: 6.737min; Method 2, retention time: 4.055 min. HRMS: m/z (M+)=502.1057(Calculated for C₂₁H₂₄F₂N₂O₆S₂=502.1044).

3-(4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazin-1-ylsulfonyl)-2,4-difluorophenol(13). ¹H NMR (400 MHz, CDCl₃) δ: 7.25 (m, 3H), 7.05 (m, 2H), 4.33 (m,4H), 3.37 (m, 4H), 3.13 (m, 4H), 1.84 (b, 1H). LC/MS: Method 1,retention time: 5.783 min; Method 2, retention time: 3.888 min. HRMS:m/z (M+)=476.0542 (Calculated for C₁₈H₁₈F₂N₂O₇S₂=476.0523).

1-(2,4-difluorophenylsulfonyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazine(14). ¹H NMR (400 MHz, DMSO-d₆) δ: 7.81 (m, 1H), 7.57 (ddd, 1H, J=10.96,9.00, 2.35 Hz) 7.32 (td, 1H, J=8.51, 2.15 Hz), 7.23-7.11 (m, 2H),7.11-7.01 (m, 1H), 4.40-4.27 (m, 4H), 3.22-3.08 (m, 4H), 3.03-2.80 (m,4H), LC/MS: Method 1, retention time: 5.910 min; Method 2, retentiontime: 3.910 min. HRMS: m/z (M+)=460.0585 (Calculated forC₁₈H₁₈F₂N₂O₆S₂=460.0574).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(phenylsulfonyl)piperazine(15). ¹H NMR (400 MHz, DMSO-d₆) δ: 7.73-7.64 (m, 3H), 7.59 (m, 2H),7.13-7.07 (m, 2H), 7.01 (m, 1H) 4.30 (m, 4H), 2.98-2.88 (m, 8H). LC/MS:Method 1, retention time: 5.706 min; Method 2, retention time: 3.883min. HRMS: m/z (M+)=424.0769 (Calculated for C₁₈H₂₀N₂O₆S₂=424.0763).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(3-(trifluoromethyl)phenylsulfonyl)piperazine(16). ¹H NMR (400 MHz, CDCl₃) δ: 7.96 (s, 1H), 7.89 (m, 2H), 7.70 (m,1H), 7.20 (m, 2H), 6.96 (d, 1H, J=8.61 Hz), 4.31 (m, 4H), 3.11 (m, 8H).LC/MS: Method 1, retention time: 6.249 min; Method 2, retention time:3.920 min. HRMS: m/z (M+)=492.0654 (Calculated forC₁₉H₁₉F₃N₂O₆S₂=492.0637).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(3-methoxyphenylsulfonyl)piperazine(17). ¹H NMR (400 MHz, DMSO-d₆) δ: 7.50 (m, 1H), 7.24 (m, 2H), 7.09 (m,3H), 7.01 (m, 1H) 4.31 (m, 4H), 3.80 (s, 3H), 2.99 (m, 4H), 2.89 (m,4H). LC/MS: Method 1, retention time: 5.819 min; Method 2, retentiontime: 3.902 min. HRMS: m/z (M+)=454.0878 (Calculated forC₁₉H₂₂N₂O₇S₂=454.0868).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(pyridin-2-ylsulfonyl)piperazine(18). ¹H NMR (400 MHz, CDCl₃) δ: 8.68 (d, 1H, J=4.7 Hz), 7.92 (m, 2H),7.51 (m, 1H), 7.24 (m, 2H), 6.99 (d, 1H, J=8.61 Hz), 4.33 (m, 4H), 3.44(m, 4H), 3.09 (m, 4H). LC/MS: Method 1, retention time: 5.205 min;Method 2, retention time: 3.772 min. HRMS: m/z (M+)=425.0720 (Calculatedfor C₁₇H₁₉N₃O₆S₂=425.0715).

2-(4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazin-1-ylsulfonyl)pyridine1-oxide (19). ¹H NMR (400 MHz, DMSO-d₆) δ: 8.27 (m, 1H), 7.90 (m, 1H),7.57 (m, 1H), 7.41 (m, 1H), 7.06 (m, 3H), 4.30 (m, 4H), 3.40 (m, 4H),2.87 (m, 4H). LC/MS: Method 1, retention time: 4.618 min; Method 2,retention time: 3.630 min. HRMS: m/z (M+)=441.0669 (Calculated forC₁₇H₁₉N₃O₇S₂=441.0664).

4-(2,6-difluorophenylsulfonyl)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperidine(20). ¹H NMR (400 MHz, CDCl₃) δ: 7.63 (m, 1H), 7.24 (m, 2H), 7.06 (t,2H, J=8.61 Hz), 6.96 (d, 1H, J=8.61 Hz), 4.31 (m, 4H), 3.88 (d, 2H,J=12.1 Hz), 3.08 (m, 1H), 2.40 (td, 2H, J=11.93, 2.35 Hz), 2.14 (m, 2H).1.96 (m, 2H). LC/MS: Method 1, retention time: 5.561 min; Method 2,retention time: 3.847 min. HRMS: m/z (M+)=459.0634 (Calculated forC₁₉H₁₉F₂NO₆S₂=459.0622).

1-(2,6-difluorophenylsulfonyl)-4-(4-methoxyphenylsulfonyl)piperazine(21). ¹H NMR (400 MHz, CDCl₃) δ: 7.66 (m, 2H), 7.54 (m, 2H), 7.02 (m,3H), 3.88 (s, 3H), 3.35 (m, 4H), 3.09 (m, 4H). LC/MS: Method 1,retention time: 5.829 min; Method 2, retention time: 3.904 min. HRMS:m/z (M+)=432.0633 (Calculated for C₁₇H₁₈F₂N₂O₅S₂=432.0625).

1,4-bis(2,6-difluorophenylsulfonyl)piperazine (22). ¹H NMR (400 MHz,CDCl₃) δ: 7.54 (m, 2H), 7.04 (t, 4H, J=12 Hz), 3.40 (s, 8H). LC/MS:Method 1, retention time: 5.851 min; Method 2, retention time: 3.911min. HRMS: m/z (M+)=438.0331 (Calculated for C₁₆H₁₄F₄N₂O₄S₂=438.034).

1-(2,6-difluorophenylsulfonyl)-4-(3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-ylsulfonyl)piperazine(23). ¹H NMR (400 MHz, DMSO-d₆) δ: 7.72 (m, 1H), 7.31-7.18 (m, 4H), 7.11(d, 1H, J=8.4 Hz), 4.22 (dt, J=17.8 Hz, 4 Hz), 3.18 (m, 4H), 2.98 (m,4H), 2.14 (m, 2H). LC/MS: Method 1, retention time: 5.973 min; Method 2,retention time: 3.925 min. HRMS: m/z (M+)=474.0747 (Calculated forC₁₉H₂₀F₂N₂O₆S₂=474.0731).

1-(benzo[d][1,3]dioxol-5-ylsulfonyl)-4-(2,6-difluorophenylsulfonyl)piperazine(24). ¹H NMR (400 MHz, DMSO-d₆) δ: 7.75 (m, 1H), 7.29 (m, 2H), 7.22 (m,1H), 7.16 (m, 1H), 7.07 (d, 1H, J=8.2 Hz), 6.17 (s, 2H), 3.17 (m, 4H),2.99 (m, 4H). LC/MS: Method 1, retention time: 5.741 min; Method 2,retention time: 3.879 min. HRMS: m/z (M+)=446.0427 (Calculated forC₁₇H₁₆F₂N₂O₆S₂=446.0418).

6-(4-(2,6-difluorophenylsulfonyl)piperazin-1-ylsulfonyl)-4-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine(25). ¹H NMR (400 MHz, DMSO-d₆) δ: 7.74 (m, 1H), 7.29 (m, 2H), 6.89-6.76(m, 3H), 4.27 (m, 2H), 3.28 (m, 2H), 3.17 (m, 4H), 2.96 (m, 4H), 2.84(s, 3H). LC/MS: Method 1, retention time: 5.514 min; Method 2, retentiontime: 3.813 min. HRMS: m/z (M+)=473.0897 (Calculated forC₁₉H₂₁F₂N₃O₅S₂=473.0891).

1-(2,6-difluorophenylsulfonyl)-4-(naphthalen-2-ylsulfonyl)piperazine(26). ¹H NMR (400 MHz, DMSO-d₆) δ: 8.39 (s, 1H), 8.18-8.03 (m, 3H),7.76-7.56 (m, 4H), 7.14 (m, 2H), 3.20-3.17 (m, 8H). LC/MS: Method 1,retention time: 5.532 min; Method 2, retention time: 3.814 min. HRMS:m/z (M+)=452.0673 (Calculated for C₂₀H₁₈F₂N₂O₄S₂=452.0676).

1-(2,6-difluorophenylsulfonyl)-4-(2,2-dimethylchroman-6-ylsulfonyl)piperazine(27). ¹H NMR (400 MHz, CDCl₃) δ: 7.54 (m, 1H), 7.43 (m, 2H), 7.03 (m,2H), 6.5 (d, 1H, J=8.4 Hz), 3.36 (m, 4H), 3.11 (m, 4H), 2.81 (m, 2H),1.84 (m, 2H), 1.36 (s, 6H). LC/MS: Method 1, retention time: 5.514 min;Method 2, retention time: 3.811 min. HRMS: m/z (M+)=486.1100 (Calculatedfor C₂₁H₂₄F₂N₂O₅S₂=486.1095).

5-(4-(2,6-difluorophenylsulfonyl)piperazin-1-ylsulfonyl)-1-methyl-1H-indole(28). ¹H NMR (400 MHz, DMSO-d₆) δ: 7.94 (s, 1H), 7.62 (m, 2H), 7.54 (d,1H, J=3.1 Hz), 7.42 (m, 1H), 7.19 (t, 2H, J=9.0 Hz), 6.63 (d, 1H, J=2.9Hz), 3.85 (s, 3H), 3.15 (m, 4H), 2.95 (m, 4H). LC/MS: Method 1,retention time: 5.893 min; Method 2, retention time: 3.914 min. HRMS:m/z (M+)=455.0793 (Calculated for C₁₉H₁₉F₂N₃O₄S₂=455.0785).

5-(4-(2,6-difluorophenylsulfonyl)piperazin-1-ylsulfonyl)-2-methylbenzo[d]thiazole(29). ¹H NMR (400 MHz, DMSO-d₆) δ: 8.51 (s, 1H), 8.07 (m, 1H), 7.73 (m,1H), 7.64 (m, 1H), 7.20 (m, 2H), 3.09 (m, 8H), 2.86 (s, 3H). LC/MS:Method 1, retention time: 5.729 min; Method 2, retention time: 3.882min; HRMS: m/z (M+)=473.0353 (Calculated for C₁₈H₁₇F₂N₃O₄S₃=473.0349).

1-(2,6-difluorophenylsulfonyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperidine(30). ¹H NMR (400 MHz, CDCl₃) δ: 7.52 (m, 1H), 7.35-7.26 (m, 2H),7.07-6.97 (m, 3H), 4.40-4.28 (m, 4H), 4.08-4.00 (m, 2H), 2.92 (m, 1H),2.66 (t, 2H, J=11.93 Hz), 2.17-2.08 (m, 2H), 1.80-1.67 (m, 2H). LC/MS:Method 1, retention time: 5.584 min; Method 2, retention time: 3.853min; HRMS: m/z (M+)=459.0631 (Calculated for C₁₉H₁₉F₂NO₆S₂=459.0622).

1-(2,6-difluorophenylsulfonyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-1,4-diazepane(31). ¹H NMR (400 MHz, CDCl₃) δ: 7.50 (m, 1H), 7.28 (m, 2H), 7.02 (m,2H), 6.96 (d, 1H, J=8.6 Hz), 4.32 (m, 4H), 3.56 (m, 4H), 3.41 (m, 4H),2.05 (m, 2H). LC/MS: Method 1, retention time: 5.812 min; Method 2,retention time: 3.891 min. HRMS: m/z (M+)=474.0731 (Calculated forC₁₉H₂₀F₂N₂O₆S₂=474.0731).

N-(2-(2,6-difluorophenylsulfonamido)ethyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide(32). ¹H NMR (400 MHz, CDCl₃) δ: 7.54 (m, 1H), 7.35 (m, 2H), 7.06 (m,2H), 6.96 (d, 1H, J=8.2 Hz), 5.37 (b, 1H), 4.73 (b, 1H), 4.31 (m, 4H),3.25 (m, 2H), 3.14 (m, 2H). LC/MS: Method 1, retention time: 4.986 min;Method 2, retention time: 3.711 min. HRMS: m/z (M+)=434.0434 (Calculatedfor C₁₆H₁₆F₂N₂O₆S₂=434.0418).

N-(3-(2,6-difluorophenylsulfonamido)propyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide(33). ¹H NMR (400 MHz, CDCl₃) δ: 7.52 (m, 1H), 7.34 (m, 2H), 7.04 (m,2H), 6.96 (d, 1H, J=8.22 Hz), 5.43 (t, 1H, J=6.46 Hz), 4.85 (b, 1H),4.31 (m, 4H), 3.21 (q, 2H, J=6.26 Hz), 3.05 (t, 2H, J=6.06 Hz), 1.74 (m,2H). LC/MS: Method 1, retention time: 5.115 min; Method 2, retentiontime: 3.730 min. HRMS: m/z (M+)=448.0571 (Calculated forC₁₇H₁₈F₂N₂O₆S₂=448.0574).

N-(4-(2,6-difluorophenylsulfonamido)butyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide(34). ¹H NMR (400 MHz, CDCl₃) δ: 7.49 (m, 1H), 7.31 (m, 2H), 7.02 (m,2H), 6.91 (d, 1H, J=8.22 Hz), 5.03 (m, 1H), 4.47 (m, 1H), 4.28 (m, 4H),3.06 (m, 2H), 2.89 (m, 2H), 1.54 (m, 4H). LC/MS: Method 1, retentiontime: 5.238 min; Method 2, retention time: 3.757 min. HRMS: m/z(M+)=462.0739 (Calculated for C₁₈H₂₀F₂N₂O₆S₂=462.0731).

N-(5-(2,6-difluorophenylsulfonamido)pentyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide(35). ¹H NMR (400 MHz, CDCl₃) δ: 7.52 (m, 1H), 7.35 (m, 2H), 7.04 (m,2H), 6.96 (d, 1H, J=8.61 Hz), 5.00 (b, 1H), 4.32 (m, 4H), 3.07 (q, 1H,J=6.65 Hz), 2.91 (t, 1H, J=6.85 Hz), 2.70 (b, 1H), 1.50 (m, 4H), 1.32(m, 2H). LC/MS: Method 1, retention time: 5.450 min; Method 2, retentiontime: 3.798 min. HRMS: m/z (M+)=476.0899 (Calculated forC₁₉H₂₂F₂N₂O₆S₂=476.0877).

N-(6-(2,6-difluorophenylsulfonamido)hexyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide(36). ¹H NMR (400 MHz, CDCl₃) δ: 7.52 (m, 1H), 7.36 (m, 2H), 7.04 (m,2H), 6.96 (d, 1H, J=8.61 Hz), 4.99 (b, 1H), 4.32 (m, 4H), 3.08 (m, 2H),2.91 (m, 2H), 1.72 (b, 1H), 1.47 (m, 4H), 1.27 (m, 4H). LC/MS: Method 1,retention time: 5.629 min; Method 2, retention time: 3.836 min. HRMS:m/z (M+)=490.1056 (Calculated for C₂₀H₂₄F₂N₂O₆S₂=490.1044).

N-((trans)-4-(2,6-difluorophenylsulfonamido)cyclohexyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide(37). ¹H NMR (400 MHz, CDCl₃) δ: 7.49 (m, 1H), 7.30 (m, 2H), 7.00 (m,2H), 6.91 (d, 1H, J=8.61 Hz), 4.95 (m, 1H), 4.47 (m, 1H), 4.28 (m, 4H),3.25 (b, 1H), 3.00 (b, 1H), 1.84 (m, 4H), 1.24 (m, 4H). LC/MS: Method 1,retention time: 5.290 min; Method 2, retention time: 3.760 min. HRMS:m/z (M+)=488.0895 (Calculated for C₂₀H₂₂F₂N₂O₆S₂=488.0887).

N-((cis)-4-(2,6-difluorophenylsulfonamido)cyclohexyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide(38). ¹H NMR (400 MHz, CDCl₃) δ: 7.49 (m, 1H), 7.35 (m, 2H), 7.00 (m,2H), 6.90 (d, 1H, J=8.61 Hz), 5.21 (m, 1H), 4.85 (m, 1H), 4.29 (m, 4H),3.42 (b, 1H), 3.20 (b, 1H), 1.45-1.65 (m, 8H). LC/MS: Method 1,retention time: 5.507 min; Method 2, retention time: 3.803 min. HRMS:m/z (M+)=488.0885 (Calculated for C₂₀H₂₂F₂N₂O₆S₂=488.0887).

N-(1-(2,6-difluorophenylsulfonyl)piperidin-4-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide(39). ¹H NMR (CDCl₃) δ: 7.50 (m, 1H), 7.33 (m, 2H), 7.00 (m, 2H), 6.93(d, 1H, J=8.61 Hz), 4.86 (d, 1H, J=6.65 Hz), 4.30 (m, 4H), 3.67 (m, 2H),3.22 (m, 1H), 2.83 (t, 2H, J=10.37 Hz), 1.86 (m, 2H), 1.56 (m, 2H).LC/MS: Method 1, retention time: 5.514 min; Method 2, retention time:3.825 min. HRMS: m/z (M+)=474.0744 (Calculated forC₁₉H₂₀F₂N₂O₆S₂=474.0731)

N-(1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperidin-4-yl)-2,6-difluorobenzenesulfonamide(40). ¹H NMR (400 MHz, CDCl₃) δ: 7.50 (m, 1H), 7.31 (m, 2H), 7.01 (m,2H), 6.96 (d, 1H, J=8.6 Hz), 4.96 (d, 1H, J=6.65 Hz), 4.37 (m, 4H), 3.64(m, 2H), 3.20 (m, 1H), 2.80 (t, 2H, J=10.4 Hz), 1.89 (m, 2H), 1.55 (m,2H). LC/MS: Method 1, retention time: 5.511 min; Method 2, retentiontime: 3.825 min. HRMS: m/z (M+)=474.0733 (Calculated forC₁₉H₂₀F₂N₂O₆S₂=474.0731).

N-(1-(2,6-difluorophenylsulfonyl)pyrrolidin-3-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide(41). ¹H NMR (400 MHz, CDCl₃) δ: 7.52 (m, 1H), 7.33 (m, 2H), 7.03 (m,2H), 6.96 (d, 1H, J=8.6 Hz), 4.85 (b, 1H), 4.32 (m, 4H), 3.84 (m, 1H),3.53 (m, 2H), 3.42 (m, 1H), 3.19 (q, 1H, J=4.7 Hz), 2.11 (m, 1H), 1.87(m, 1H). LC/MS: Method 1, retention time: 5.339 min; Method 2, retentiontime: 3.789 min. HRMS: m/z (M+)=460.0578 (Calculated forC₁₈H₁₈F₂N₂O₆S₂=460.0574).

N-(1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)pyrrolidin-3-yl)-2,6-difluorobenzenesulfonamide(42). ¹H NMR (400 MHz, CDCl₃) δ: 7.51 (m, 1H), 7.33 (m, 2H), 7.03 (m,2H), 6.95 (d, 1H, J=8.6 Hz), 5.02 (b, 1H), 4.31 (m, 4H), 3.88 (m, 1H),3.59 (m, 2H), 3.44 (m, 1H), 3.16 (q, 1H, J=4.7 Hz), 2.08 (m, 1H), 1.88(m, 1H). LC/MS: Method 1, retention time: 5.339 min; Method 2, retentiontime: 3.792 min. HRMS: m/z (M+)=460.0587 (Calculated forC₁₈H₁₈F₂N₂O₆S₂=460.0574).

N-((1-(2,6-difluorophenylsulfonyl)azetidin-3-yl)methyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide(43). ¹H NMR (400 MHz, CDCl₃) δ: 7.53 (m, 1H), 7.30 (m, 2H), 7.05 (m,2H), 6.94 (d, 1H, J=8.6 Hz), 4.40 (m, 1H) 4.30 (m, 4H), 4.04 (t, 2H,J=8.2 Hz), 3.66 (dd, 2H, J=8.4, 5.65 Hz), 3.08 (t, 2H, J=6.7 Hz), 2.69(m, 1H). LC/MS: Method 1, retention time: 5.295 min; Method 2, retentiontime: 3.780 min. HRMS: m/z (M+)=460.0582 (Calculated forC₁₈H₁₈F₂N₂O₆S₂=460.0574).

N-((1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)azetidin-3-yl)methyl)-2,6-difluorobenzenesulfonamide(44). ¹H NMR (400 MHz, CDCl₃) δ: 7.51 (m, 1H), 7.27 (m, 2H), 7.00 (m,3H), 5.23 (t, 1H, J=6.06 Hz), 4.31 (m, 4H), 3.78 (t, 2H, J=8.22 Hz),3.47 (dd, 2H, J=8.41, 5.67 Hz), 3.10 (t, 2H, J=6.7 Hz), 2.62 (m, 1H).LC/MS: Method 1, retention time: 5.234 min; Method 2, retention time:3.767 min. HRMS: m/z (M+)=460.0583 (Calculated forC₁₈H₁₈F₂N₂O₆S₂=460.0574).

(S)-4-(2,6-difluorophenylsulfonyl)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-2-methylpiperazine(45). ¹H NMR (400 Hz, CDCl₃) δ: 7.55 (m, 1H), 7.26 (m, 2H), 7.04 (m,2H), 6.92 (m, 1H), 4.30 (m, 4H), 4.21 (m, 1H), 3.84 (d, 1H, J=12.1 Hz),3.72 (d, 1H, J=12.9 Hz), 3.61 (d, 1H, J=12.1 Hz), 3.24 (td, J=12.5, 3.13Hz), 2.86 (dd, 1H, J=12.1, 2.74 Hz), 2.72 (td, 1H, J=11.9, 3.1 Hz), 1.13(d, 3H, J=6.7 Hz). LC/MS: Method 1, retention time: 5.873 min; Method 2,retention time: 3.905 min. HRMS: m/z (M+)=474.0736 (Calculated forC₁₉H₂₀F₂N₂O₆S₂=474.0731).

(R)-4-(2,6-difluorophenylsulfonyl)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-2-methylpiperazine(46). ¹H NMR (400 MHz, CDCl₃) δ: 7.55 (m, 1H), 7.26 (m, 2H), 7.04 (m,2H), 6.92 (m, 1H), 4.30 (m, 4H), 4.21 (m, 1H), 3.84 (d, 1H, J=12.1 Hz),3.72 (d, 1H, J=12.9 Hz), 3.61 (d, 1H, J=12.1 Hz), 3.24 (td, 1H, J=12.5,3.1 Hz), 2.86 (dd, 1H, J₁=12.1, 2.7 Hz), 2.72 (td, 1H, J=11.9, 13.0 Hz),1.13 (d, 3H, J=6.7 Hz). LC/MS: Method 1, retention time: 5.872 min;Method 2, retention time: 3.905 min. HRMS: m/z (M+)=474.0736 (Calculatedfor C₁₉H₂₀F₂N₂O₆S₂=474.0731).

(S)-1-(2,6-difluorophenylsulfonyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-2-methylpiperazine(48). ¹H NMR (400 MHz, CDCl₃) δ: 7.50 (m, 1H), 7.22 (m, 2H), 7.00 (m,3H), 4.33 (m, 5H), 3.92 (d, 1H, J=13.7 Hz), 3.70 (d, 1H, J=11.4 Hz),3.50 (d, 1H, J=11.4 Hz), 3.38 (m, 1H), 2.53 (dd, 1H, J=11.4, 3.5 Hz),2.39 (td, J=11.8, 3.3 Hz), 1.22 (d, 3H, J=7.0 Hz). LC/MS: Method 1,retention time: 5.912 min; Method 2, retention time: 3.910 min. HRMS:m/z (M+)=474.0726 (Calculated for C₁₉H₂₀F₂N₂O₆S₂=474.0731).

(R)-1-(2,6-difluorophenylsulfonyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-2-methylpiperazine(47). ¹H NMR (400 MHz, CDCl₃) δ: 7.50 (m, 1H), 7.22 (m, 2H), 7.00 (m,3H), 4.33 (m, 5H), 3.92 (d, 1H, J=13.7 Hz), 3.70 (d, 1H, J=11.4 Hz),3.50 (d, 1H, J=11.4 Hz), 3.38 (m, 1H), 2.53 (dd, 1H, J=11.4, 3.5 Hz),2.39 (td, 1H, J=11.8, 3.3 Hz), 1.22 (d, 3H, J=7.0 Hz). LC/MS: Method 1,retention time: 5.910 min; Method 2, retention time: 3.912 min. HRMS:m/z (M+)=474.0727 (Calculated for C₁₉H₂₀F₂N₂O₆S₂=474.0731).

Synthesis of oxo-piperazine Derivatives 49 and 50

Exemplified by 49

Method A was used to introduce the 2,6-difluorosulfonyl group.

4-(2,6-difluorophenylsulfonyl)piperazin-2-one (500 mg, 1.81 mmol, 1equiv.) was dissolved in THF (5 mL) and cooled to −78° C. LHMDS (1.85 mLof 1.0 M THF solution, 1.9 mmol, 1.05 equiv.) was then added dropwiseand the solution stirred at −78° C. for 1 h. A solution of2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride (510 mg, 2.17 mmol,1.2 equiv.) in THF (2 mL) was then added drop-wise to the cold solution.The reaction was stirred at −78° C. for 15 minutes then allowed to warmto room temperature and stirred an additional 1 h. The reaction wascarefully quenched with saturated aqueous ammonium chloride (˜5 mL), anddiluted with ethyl acetate (˜15 mL). The organic layer was washed twicewith saturated aqueous ammonium chloride, once with brine, dried oversodium sulfate and concentrated. The residue was dissolved in DMSO andpurified by reverse phase HPLC.

4-(2,6-difluorophenylsulfonyl)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazin-2-one(49). ¹H NMR (400 Hz, CDCl₃) δ: 7.58 (m, 1H), 7.29 (m, 3H), 7.03 (m,2H), 4.31 (m, 4H), 4.07 (m, 2H), 3.81 (s, 2H), 3.47 (m, 2H). LC/MS:Method 1, retention time: 5.631 min; Method 2, retention time: 3.858min. HRMS: m/z (M+)=474.0372 (Calculated for C₁₈H₁₆F₂N₂O₇S₂=474.0367).

1-(2,6-difluorophenylsulfonyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazin-2-one(50). ¹H NMR (400 MHz, CDCl₃) δ: 7.58 (m, 1H), 7.29 (m, 3H), 7.03 (m,2H), 4.33 (m, 4H), 4.07 (m, 2H), 3.78 (s, 2H), 3.44 (m, 2H). LC/MS:Method 1, retention time: 5.612 min; Method 2, retention time: 3.849min. HRMS: m/z (M+)=474.0366 (Calculated for C₁₈H₁₆F₂N₂O₇S₂=474.0367).

Compounds of formula II were prepared as follows:

Ethyl 2-azido-3-(5-bromothiophen-2-yl)acrylate (60). A solution ofsodium (2.76 g, 120 mmol) in absolute EtOH (120 mL) was cooled in anice-bath and a mixture of 5-bromo-2-formylthiophene (5.73 g, 30 mmol)and ethyl azidoacetate (15.49 g, 120 mmol) was added dropwise during 30min period. The bath was removed and the reaction mixture was stirred atroom temperature for another 30 min. A cold solution of saturatedaqueous NH₄Cl solution (100 mL) was added and the resulting solution wasextracted with diethyl ether (3×100 mL) and the combined organic layerswere washed with brine (200 mL), dried over Na₂SO₄. After removingdiethyl ether under reduced pressure, the crude product was purified bycolumn chromatography (EtOAc/Hexane: 1/50) to give acrylate 60 (3.81 g,42%) as a light yellow solid.

Ethyl 2-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylate (61). Acrylate 60(3.81 g, 12.6 mmol) in o-xylene was refluxed for 20 min. After removingthe o-xylene, the crude product was purified by column chromatography(EtOAc/Hexane: 1/10) to give carboxylate 61 (2.83 g, 82%) as a whitesolid.

Ethyl 2-bromo-6-formyl-4H-thieno[3,2-b]pyrrole-5-carboxylate (62). ToDMF (2.05 mL) cooled by ice/water was added POCl₃ dropwise and themixture was stirred for 30 min. A solution of carboxylate 61 (1.96 g,7.15 mmol) in DMF (2.5 mL) was added at this temperature and the mixturewas allowed to warm to room temperature then heated to 60° C. After 16h, the reaction mixture was cooled to room temperature. and poured intoice/water. The mixture was extracted with EtOAc (3×20 mL) and thecombined organic layers were washed with aqueous saturated NaHCO₃ andbrine, dried over Na₂SO₄. After removed the organic solvent, the residuewas purified by column chromatography (EtOAc/Hexane: 1/4) to give thedesired aldehyde 62 (1.62 g, 75%) as a white solid.

Ethyl 2-bromo-6-formyl-4-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylate(63). To a solution of aldehyde 62 (515 mg, 1.70 mmol) in DMF (5 mL) wasadded potassium carbonate (707 mg, 5.12 mmol) and iodomethane (0.27 mL,3.40 mmol) and the resulting mixture was stirred at room temperature for2 hr. To the mixture was added H₂O (30 mL) and EtOAc (50 mL) and theorganic layer was separated and washed with brine and dried over Na₂SO₄.After removing the organic solvent under reduced pressure, the residuewas purified by column chromatography (EtOAc/Hexane: 1/8) to give thedesired N-methylated product 63 (440 mg, 82%) as a white solid.

2-Bromo-4-methyl-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone (64). TheN-methylated product 63 (420 mg, 1.33 mmol) was dissolved in warm2-ethoxyethanol (26 mL). To a refluxed solution of hydrazine monohydrate(1 mL, 31.9 mmol) and 2-ethoxyethanol (5 mL) under nitrogen was addedprepared N-methylated product 62 solution dropwise over a 2 hr periodand the solution continued to reflux for additional 1 hr. After coolingto room temperature, about half of the 2-ethoxyethanol was removed underreduced pressure and the remaining solution was refrigerated at −20° C.overnight. The precipitate was filtered and washed with 2-ethoxyethanolto give desired pyridazinone 64 (354 mg, 94%) as a white solid.

2-Bromo-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(65). To a solution of pyridazinone 63 (354 mg, 1.25 mmol) in EtOH (5mL) was added potassium carbonate (1.02 g, 7.35 mmol) and 2-fluorobenzylbromide (0.94, 4.98) and the resulting mixture was heated at 60° C. for2 hr. After cooling to room temperature, to the mixture H₂O (20 mL) andEtOAc (50 mL) was added and the organic layer was separated and washedwith brine and dried over Na₂SO₄. After removing the solvent underreduced pressure, the residue was purified by column chromatography(EtOAc/Hexane: 1/4) to give desired pyridazinone 65 (371 mg, 76%) as awhite solid. This compound was used as a versatile intermediateunderwent a variety of transformations as described below.

The procedure for the synthesis of this lead compound is the same aspyridazinone 65. We also developed a more efficient, generalizedprocedure for the last step of coupling the pyridazinone 64 with2-fluorobenzyl bromide. This procedure was adopted for the syntheses ofanalogue 89-92, 102 and 103-121.

2,4-Methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(NCGC00031955) (66). To a solution of2,4-dimethyl-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone (20 mg, 0.086mmol) in DMF (0.5 mL) was added potassium tert-butoxide (1.5 eq.) and2-fluorobenzyl bromide (2 eq.) and the mixture was stirred for 1 h atroom temperature. The mixture was filtered through a frit attached to asyringe and washed with DMF and the total filtrate was 2 mL. The DMFsolution was directly subjected to purification by preparative HPLC togive the desired product as a white solid. For other analogues preparedin this way, the yield ranges from 35% to 90%. ¹H NMR (400 MHz, CDCl₃) δ8.20 (s, 1H), 7.26-7.19 (m, 2H), 7.09-7.02 (m, 2H), 6.92 (q, 1H, J=1.2Hz), 5.53 (s, 2H), 4.27 (s, 3H), 2.64 (d, 3H, J=1.2 Hz); LC/MS: Method1, retention time: 6.313 min; Method 2, retention time: 3.992 min; HRMS:m/z (M+H⁺)=328.0925 (Calculated for C₁₇H₁₅FN₃OS=328.0920).

2-Vinyl-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(67). To a microwave vessel was added bromide 65 (24 mg, 0.06 mmol),vinyl boronic acid pinacol ester (28 mg, 0.18 mmol), Pd(PPh₃)₂Cl₂ (8.4mg, 0.012 mmol, 20 mol %), 1M aqueous Na₂CO₃ solution (0.16 mL) andCH₃CN (0.16 mL). The mixture was purged with nitrogen for 1 min and thevessel was capped. The vessel was subjected to be microwaved at 120° C.for 20 min. After cooling down, the cap was removed and the mixture waspartitioned in EtOAc (10 mL) and H₂O (5 mL). The organic layer wasseparated, washed with brine and dried (MgSO₄). After removing EtOAcunder reduced pressure, the crude product was directly purified bypreparative TLC (EtOAc/Hexane: 1/4) to give alkene 67 (16 mg, 75%) as awhite solid.

2-Ethyl-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(68). To a solution of compound 67 (10 mg, 0.32 mmol) in MeOH (1 mL) wasadded 10 wt % Pd/C (5 mg) and stirred under H₂ (1 atm) for 2 h. Thecatalyst was filtered and MeOH was removed under reduced pressure togive a residue which was purified by column chromatography(EtOAc/Hexane: 1/4) to give the desired product 68 (8 mg, 80%) as awhite solid. ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.24-7.17 (m, 2H),7.07-7.00 (m, 2H), 6.80 (t, 1H, J=1.0 Hz), 5.51 (s, 2H), 4.26 (s, 3H),2.95 (qd, 2H, J=7.8, 1.0 Hz), 1.37 (t, 3H, J=7.8 Hz); LC/MS: Method 1,retention time: 6.658 min; Method 2, retention time: 4.052 min; HRMS:m/z (M+H⁺)=342.1073 (Calculated for C₁₈H₁₇FN₃OS=342.1076).

2-Isopropyl-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(69). Analogue 69 was prepared in the same procedure as analogue 68. ¹HNMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.28-7.17 (m, 2H), 7.07-7.00 (m,2H), 6.81 (d, 1H, J=0.8 Hz), 5.51 (s, 2H), 4.26 (s, 3H), 3.28-3.20 (m,1H), 1.39 (d, 6H, J=6.8 Hz); LC/MS: Method 1, retention time: 6.942 min;Method 2, retention time: 4.106 min; HRMS: m/z (M+H⁺)=356.1230(Calculated for C₁₉H₁₉FN₃OS=356.1233).

4-Methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(70). To a solution of 65 (20 mg, 0.051 mmol) in MeOH (5 mL) was added10 wt % Pd/C (10 mg) and stirred under H₂ (1 atm) for 1 h. The catalystwas filtered through a pad of Celite™ and MeOH was removed under reducedpressure. The residue was purified by column chromatography(EtOAc/Hexane: 1/4) to give the de-brominated product 70 (13 mg, 81%) asa white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.27 (s, 1H), 7.55 (d, 1H,J=5.2 Hz), 7.27-7.21 (m, 2H), 7.10 (d, 1H, J=5.2 Hz), 7.09-7.04 (m, 2H),5.54 (s, 2H), 4.34 (s, 3H); LC/MS: Method 1, Retention time: 5.995 min;Method 2, retention time: 3.925 min; HRMS: m/z (M+H⁺)=314.0760(Calculated for C₁₆H₁₃FN₃OS=314.0763).

2-Methoxy-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(71). To anhydrous MeOH (0.27 mL) was slowly added sodium pieces (16 mg,0.69 mmol). After ceasing to produce H₂, excess MeOH was removed underreduced pressure. To freshly prepared sodium methoxide was added 65 (34mg, 0.087 mmol), CuI (3.3 mg, 0.017 mmol) and dioxane (0.3 mL) and themixture was refluxed for 16 h. After cooling to r.t., the mixture waspartitioned in water and EtOAc and the aqueous layer was furtherextracted with EtOAc. The organic layers were washed with brine anddried over Na₂SO₄. After the removal of organic solvent, the residue wasdirectly purified by preparative HPLC to give desired methoxysubstituted analogue 71 (7 mg, 44% based on recovered starting material)and recovered starting material (16 mg). ¹H NMR (400 MHz, CDCl₃) δ 8.14(s, 1H), 7.26-7.20 (m, 2H), 7.09-7.03 (m, 2H), 6.27 (s, 1H), 5.53 (s,2H), 4.25 (s, 3H), 4.01 (s, 3H); LC/MS: Method 1, retention time: 6.169min; Method 2, retention time: 3.939 min; HRMS: m/z (M+H⁺)=344.0868(Calculated for C₁₇H₁₅FN₃O₂S=344.0869).

2-Methylthio-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(72). To a solution of 65 (50 mg, 0.13 mmol) in DMF (0.5 mL) was addedcopper (I) bromide (18 mg, 0.13 mmol) and sodium thiomethoxide (27 mg,0.38 mmol) and the mixture was heated at 140° C. for 2 h. After coolingto room temperature, the mixture was partitioned in EtOAc (10 mL) andwater (10 mL) and the organic layer was separated, washed with brine anddried over Na₂SO₄. After the removal of organic solvent, the residue waspurified by preparative HPLC to give the desired thiomethyl analogue 71(16 mg, 35%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.18 (s, 1H),7.25-7.19 (m, 2H), 7.09 (s, 1H), 7.07-7.02 (m, 2H), 5.51 (s, 2H), 4.26(s, 3H), 2.58 (s, 3H); LC/MS: Method 1, retention time: 6.570 min;Method 2, retention time: 4.063 min; HRMS: m/z (M+H⁺)=360.0637(Calculated for C₁₇H₁₅FN₃OS₂=360.0641).

2-Methylsulfinyl-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone (73) and2-Methylsulfonyl-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(74). To a solution of 7 (25 mg, 0.069 mmol) in DCM (2 mL) was addedmCPBA (1.5 eq.) and the mixture was stirred at room temperature for 2 h.The mixture was diluted with DCM (10 mL) and washed with saturatedaqueous NaHCO₃ and dried over Na₂SO₄. After the removal of solvent, thecrude product was purified by preparative HPLC to give sulfoxide 73 (10mg, 40%) and sulfone 74 (9 mg, 36%). 73: ¹H NMR (400 MHz, CDCl₃) δ 8.28(s, 1H), 7.57 (s, 1H), 7.30-7.22 (m, 2H), 7.10-7.04 (m, 2H), 5.53 (s,2H), 4.34 (s, 3H), 3.01 (s, 3H); LC/MS: Method 1, retention time, 4.967min; Method 2, retention time, 3.683 min; HRMS: m/z (M+H⁺)=376.0587(Calculated for C₁₇H₁₅FN₃O₂S₂=376.0590). 74: ¹H NMR (400 MHz, CDCl₃) δ8.28 (s, 1H), 7.80 (s, 1H), 7.32-7.23 (m, 2H), 7.11-7.04 (m, 2H), 5.53(s, 2H), 4.36 (s, 3H), 3.26 (s, 3H); LC/MS: Method 1, retention time:5.621 min; Method 2, retention time: 3.830 min; HRMS: m/z (M+H⁺)392.0537 (calculated for C₁₇H₁₅FN₃O₃S₂ ⁺) 392.0539.

Ethyl 6-formyl-2-nitro-4H-thieno[3,2-b]pyrrole-5-carboxylate (77).Carboxylate 76 was prepared in the same procedure as 62. PulverizedCu(NO₃)₂ hydrate (234 mg, 0.97 mmol) dissolved in acetic anhydride (2mL) was added dropwise to a solution of 76 (240 mg, 1.08 mmol) dissolvedin acetic anhydride (5 mL) cooled by ice/water bath. The addition wascompleted in 1.5 h and the mixture was then stirred at room temperaturefor 2 h. The salt was filtered and the filtrate was introduced intoice/water. The mixture was extracted with diethyl ether (3×10 mL) andthe combined organic layers were washed with saturated aqueous sodiumcarbonate solution and dried over MgSO₄. After the removal of organicsolvent, the residue was purified by column chromatography(EtOAc/Hexane: 1/2) to give the carboxylate 77 (236 mg, 82%) as a lightyellow solid.

2-Nitro-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(80). Methylated intermediate 78 was prepared as compound 63. To asolution of 77 (150 mg, 0.53 mmol) in EtOH (15 mL) was added hydrazine(0.4 mL, 12.72 mmol) and the solution was stirred for 30 min. Removingthe EtOH and excess hydrazine gave the desired pyridazinone 79. To asolution of 79 (133 mg, 0.53 mmol) in DMF (5 mL) was added potassiumcarbonate (439 mg, 3.18 mmol) and 2-fluorobenzyl bromide (501 mg, 2.65mmol) and the mixture was stirred at room temperature for 2 h. Themixture was partitioned in EtOAc (30 mL) and water (30 mL) and theorganic layer was separated and washed with brine and dried over Na₂SO₄.After the removal of organic solvent, the residue was directly purifiedby preparative HPLC to give the nitro substituted analogue 80 (70 mg,37%) as a light yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.27 (s, 1H),8.06 (s, 1H), 7.35-7.24 (m, 2H), 7.11-7.05 (m, 2H), 5.52 (s, 2H), 4.37(s, 3H); LC/MS: Method 1, retention time: 6.185 min; Method 2, retentiontime: 3.978 min; HRMS: m/z (M+H⁺)=359.0607 (Calculated forC₁₆H₁₂FN₄O₃S=359.0614).

2-Acetylamido-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]-pyridazinone(81). A mixture of 65 (23 mg, 0.059 mmol), CuI (2.2 mg, 0.012 mmol),K₃PO₄ (28 mg, 0.12 mmol), acetamide (14 mg, 0.24 mmol),trans-N,N′-dimethylcyclohexane-1,2-diamine (3.4 mg, 0.024 mmol) anddioxane (0.5 mL) was sealed in a tube and stirred and heated in an oilbath at 90° C. for 16 h. The mixture was partitioned between water (5mL) and DCM (5 mL). The aqueous layer was separated and furtherextracted with DCM (2×5 mL) and the combined organic layers were washedwith brine and dried over Na₂SO₄. After the removal of organic solvent,the residue was directly purified by preparative HPLC to give 81 (11 mg,51%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.21 (s, 1H), 8.01 (br.s, 1H), 7.27-7.20 (m, 2H), 7.09-7.03 (m, 2H), 6.74 (s, 1H), 5.53 (s,2H), 4.26 (s, 3H), 2.26 (s, 3H); LC/MS: Method 1, retention time: 5.186min; Method 2, retention time: 3.727 min; HRMS: m/z (M+H⁺)=371.0974(Calculated for C₁₈H₁₆FN₄O₂S=371.0978).

2-Cyano-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(82). To a solution of bromo substituted analogue 65 (20 mg, 0.051 mmol)in DMF (0.3 mL) was added CuCN (9.1 mg, 0.10 mmol, 2 eq.) and themixture was heated at 140° C. overnight under N₂ atmosphere. Aftercooling to room temperature, the mixture was filtered and washed withEtOAc (10 mL). The filtrate was washed with water, brine and dried overNa₂SO₄. After the removal of organic solvent, the residue was purifiedby preparative HPLC to give the desired cyano substituted analogue 82 (7mg, 40%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.26 (s, 1H), 7.64(s, 1H), 7.32-7.23 (m, 2H), 7.10-7.05 (m, 2H), 5.53 (s, 2H), 4.35 (s,3H); LC/MS: Method 1, retention time: 5.905 min; Method 2, retentiontime: 3.907 min; HRMS: m/z (M+H⁺)=339.0712 (Calculated forC₁₇H₁₂FN₄OS=339.0716).

Methyl4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone-2-carboxylate(83). To a solution of 65 (40 mg, 0.10 mmol) in MeOH (0.37 mL) and DMSO(0.37 mL) was added Pd(OAc)₂ (5.7 mg, 0.025 mmol),1,3-bis(diphenylphosphino)propane (dppp) (10.5 mg, 0.025 mmol) andtriethyl-amine (16 μl, 0.11 mmol) and the mixture was exchanged tocarbon monoxide (1 atm) atmosphere and heated at 65° C. overnight. Aftercooling to room temperature, the mixture was taken into EtOAc (10 mL)and washed with water and brine. The organic layer was dried overNa₂SO₄. After the removal of organic solvent, the residue was purifiedby preparative HPLC to give the carboxylate 83 (17 mg, 46%) as a whitesolid. ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.84 (s, 1H), 7.30-7.23(m, 2H), 7.11-7.04 (m, 2H), 5.54 (s, 2H), 4.34 (s, 3H), 3.96 (s, 3H);LC/MS: Method 1, retention time: 6.170 min; Method 2, retention time:3.949 min; HRMS: m/z (M+H⁺)=372.0816 (Calculated forC₁₈H₁₅FN₃O₃S=372.0818).

2-Formyl-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(84). To DMF (52 μl, 0.67 mmol) in DCE (1 mL) cooled by ice/water wasadded POCl₃ (42 μl, 0.46 mmol) and the mixture was stirred at roomtemperature for 30 min. Analogue 70 (70 mg, 0.22 mmol) in DCE (1.2 mL)was added and the mixture was refluxed for 24 h. After cooling, themixture was pour into ice/water and extracted with EtOAc (3×10 mL). Thecombined organic layers were washed with saturated aqueous NaHCO₃ anddried over Na₂SO₄. After the removal of organic solvent, the residue waspurified by column chromatography (EtOAc/Hexane: 1/3) to give 84 (19 mg,25%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 10.00 (s, 1H), 8.31 (s,1H), 7.78 (s, 1H), 7.32-7.23 (m, 2H), 7.10-7.05 (m, 2H), 5.54 (s, 2H),4.37 (s, 3H); LC/MS: Method 1, retention time: 5.734 min; Method 2,retention time: 3.878 min; HRMS: m/z (M+H⁺)=342.0708 (Calculated forC₁₇H₁₃FN₃O₂S=342.0713).

2-Hydroxylmethyl-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(85). The analogue was prepared by reducing the corresponding aldehyde84 with sodium borohydride in MeOH. The crude product was purified bycolumn chromatography (MeOH/DCM 1/10) to give 85 (10 mg, 99%) as a whitesolid. ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.30-7.22 (m, 2H),7.10-7.02 (m, 2H), 6.98 (s, 1H), 5.53 (s, 2H), 4.89 (s, 2H), 4.25 (s,3H), 2.50 (br.s, 1H); LC/MS: Method 1, retention time: 5.143 min; Method2, retention time: 3.682 min; HRMS: m/z (M+H⁺)=344.0868 (Calculated forC₁₇H₁₅FN₃O₂S=344.0869).

4-Methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone,2-ylboronic acid (86). To a solution of tetramethylethylenediamine (36mg, 0.31 mmol) in THF (1.3 mL) at 15° C. was added isopropylmagnesiumchloride (0.16 mL, 2M in THF) and the mixture was stirred for 30 min.Analogue 65 (100 mg, 0.26 mmol) was added as a solid to this mixture andstirred at room temperature for 15 min then cooled to 0° C. To themixture was added trimethylboronate (57 μl, 0.51 mmol) and the mixturewas stirred at 0° C. for another 10 min. The mixture was quenched with0.1N HCl and extracted with EtOAc (3×5 mL). The combined organic layerswere dried over MgSO₄. The organic solvent was removed and the residuewas directly purified by preparative HPLC to give the boronic acid 86(39 mg, 43%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.32 (s, 1H),7.58 (s, 1H), 7.28-7.20 (m, 2H), 7.12-7.04 (m, 2H), 5.54 (s, 2H), 4.31(s, 3H), 3.25 (s, 2H); Method 1, retention time: 5.112 min; Method 2,retention time: 3.715 min; HRMS: m/z (M+H⁺)=358.0833 (Calculated forC₁₆H₁₄BFN₃O₃S=358.0833).

2-Acetyl-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(87). To a solution of alcohol 88 (10 mg, 0.028 mmol) in DMSO (0.3 mL)was added IBX (24 mg, 0.084 mmol) and the mixture was stirred at roomtemperature for 2 h. The mixture was partitioned between EtOAc (10 mL)and saturated aqueous NaHCO₃ (5 mL). The organic layer was separated andwashed with brine and dried over MgSO₄. After the removal of organicsolvent, the residue was purified by preparative HPLC to give desiredketone (9 mg, 90%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s,1H), 7.71 (s, 1H), 7.31-7.22 (m, 2H), 7.10-7.04 (m, 2H), 5.53 (s, 2H),4.36 (s, 3H), 2.65 (s, 3H); LC/MS: Method 1, retention time: 5.383 min;Method 2, retention time: 3.888 min; HRMS: m/z (M+H⁺)=356.0868(Calculated for C₁₈H₁₅FN₃O₂S=356.0869).

2-(2-hydroxylpropyl)-4-methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole-[3,2-d]pyridazinone(88). To a solution of tetramethylethylenediamine (12 mg, 0.10 mmol) inTHF (0.4 mL) at 15° C. was added isopropylmagnesium chloride (0.05 mL,2M in THF, 0.10 mmol) and the mixture was stirred for 30 min. Bromosubstituted analogue 65 (30 mg, 0.076 mmol) was added as a solid at thistemperature and the mixture was further stirred at r.t. for 15 min thencooled to 0° C. The Grignard reagent intermediated was treated withexcess cold acetaldehyde and the mixture was stirred at 0° C. for 10min. After quenching the reaction with saturated aqueous NH₄Cl, themixture was partitioned between water (5 mL) and EtOAc (5 mL) and theaqueous layer was extracted with EtOAc (2×5 mL) and the combined organiclayer were washed with brine and dried over Na₂SO₄. After the removal oforganic solvent, the residue was purified by preparative HPLC to give 88(6.8 mg, 23%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.18 (s, 1H),7.29-7.21 (m, 2H), 7.10-7.03 (m, 2H), 6.92 (s, 1H), 5.53 (s, 2H),5.15-5.08 (m, 1H), 4.21 (s, 3H), 2.63 (d, 1H, J=6.4 Hz), 1.65 (d, 3HJ=6.4 Hz); LC/MS: Method 1, retention time: 5.381 min; Method 2,retention time: 3.769 min; HRMS: m/z (M+H⁺)=358.1024 (Calculated forC₁₈H₁₇FN₃O₂S=358.1026).

Analogues 89-91 were prepared in the same procedure as 66. For analogue89, the nitrogen on the pyrrole ring was protected with Boc, which wasremoved under acidic condition.

2-Methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(89). ¹H NMR (400 MHz, CDCl₃) δ 11.52 (br.s, 1H), 8.31 (s, 1H),7.28-7.20 (m, 2H), 7.14-7.02 (m, 2H), 6.74 (q, 1H, J=0.8 Hz), 5.65 (s,2H), 2.62 (d, 3H, J=0.8 Hz); LC/MS: Method 1, retention time: 5.540 min;Method 2, retention time: 3.806 min; HRMS: m/z (M+H⁺)=314.0761(Calculated for C₁₆H₁₃FN₃OS=314.0763)

2-Methyl-4-ethyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(90). ¹H NMR (400 MHz, CDCl₃) δ 8.24 (s, 1H), 7.26-7.20 (m, 2H),7.09-7.02 (m, 2H), 6.82 (q, 1H, J=0.8 Hz), 5.53 (s, 2H), 4.75 (q, 2H,J=7.2 Hz), 2.65 (d, 3H, 0.8 Hz), 1.48 (t, 3H, J=7.2 Hz); LC/MS: Method1, retention time: 6.630 min; Method 2, retention time: 4.052 min; HRMS:m/z (M+H⁺)=342.1075 (Calculated for C₁₈H₁₇FN₃OS₂=342.1076).

2-Methyl-4-isopropyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(91). ¹H NMR (400 MHz, CDCl₃) δ 8.22 (s, 1H), 7.26-7.19 (m, 2H),7.08-7.02 (m, 2H), 6.93 (q, 1H, J=1.0 Hz), 6.25-6.14 (m, 1H), 5.53 (s,2H), 2.65 (d, 3H, J=1.0 Hz), 1.58 (d, 6H, J=7.2 Hz); LC/MS: Method 1,retention time: 6.897 min; Method 2, retention time: 4.100 min; HRMS:m/z (M+H⁺)=356.1232 (Calculated for C₁₉H₁₉FN₃OS₂=356.1233).

2,4,8-Methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]-pyridazinone(92). To a solution of carboxylate 79 (251 mg, 1.00 mmol) in THF (5 mL)cooled to −78° C. was added 3M MeMgCl solution in THF (0.33 mL, 1 mmol).After work-up, the crude product was purified by column chromatography(EtOAc/Hexane: 1/2) to give the desired secondary alcohol 93 (135 mg,54%) as a white solid. The secondary alcohol was oxidized using IBX tothe methyl ketone 94 (95%) in the same procedure as preparing analogue87. Following the same procedure for preparing 66, analogue 92 wasobtained. ¹H NMR (400 MHz, CDCl₃) δ 7.26-7.16 (m, 2H), 7.08-7.01 (m,2H), 6.82 (q, 1H, J=1.0 Hz), 5.49 (s, 2H), 4.27 (s, 3H), 2.65 (d, 3H,J=1.0 Hz), 2.56 (s, 3H); LC/MS: Method 1, retention time: 6.659 min;Method 2, retention time: 3.762 min; HRMS: m/z (M+H⁺)=342.1077(Calculated for C₁₈H₁₇FN₃OS=342.1076).

Ethyl-6-nitro-4H-thieno[3,2-b]pyrrole-5-carboxylate (95). PulverizedCu(NO₃)₂ hydrate (953 mg, 4.10 mmol) dissolved in acetic anhydride (8.2mL) was added dropwise to a solution of carboxylate 96 dissolved inacetic anhydride (10 mL) at 0° C. The addition was completed in 1.5 hand the mixture was then stirred at r.t. for 2 h. After filtration theorganic layer was poured over ice and extracted with diethyl ether (3×30mL). The combined organic layers were washed with saturated aqueoussodium carbonate solution and dried over MgSO₄. After the removal oforganic solvent, the residue was purified by column chromatography(EtOAc/Hexane: 1/6) to give the desired nitration product 94 (148 mg,12%) as a light yellow solid along with another nitration product 95(300 mg, 25%).

Ethyl-6-amino-4-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylate (98).Compound 94 was treated with iodomethane and potassium carbonate to givethe N-methylated product 97. To 97 (60 mg, 0.24 mmol) in EtOH (1.3 mL)was added tin(II) chloride (358 mg, 1.89 mmol). Concentrated HCl (1.3mL) was added dropwise at 0° C. and the mixture was heated at 35° C. for2 h. LC/MS found the reaction was completed and formed the desiredamine. After the mixture was cooled to room temperature, neutralized themixture to pH=9 with 1N aqueous NaOH solution and extracted with EtOAc.The combined extracts were washed with brine and dried over Na₂SO₄.After the removal of organic solvent, the crude product of amine 98 (49mg) was directly used for the next step.

4-Methyl-4H-thieno[3,2-b]pyrrole[3,2-d]pyrimidinone (99). To amine 98(49 mg, 0.22 mmol) was added ammonium formate (22 mg, 0.35 mmol) andformamide (0.3 mL) and the mixture was heated at 120° C. in sealed tubefor 16 h. After cooling to room temperature, the mixture was poured intoice/water and extracted with EtOAc (3×10 mL) and the combined organiclayers were washed with brine and dried over Na₂SO₄. Removing thesolvent afforded the crude product pyrimidinone 99 (40 mg) which wasdirectly used for the next step.

4-Methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyrimidinone(100). To pyrimidinone 99 (40 mg, 0.20 mmol) in EtOH (2 mL) was addedpotassium carbonate (38 mg, 0.27 mmol) and 2-fluorobenzyl bromide (44mg, 0.24 mmol) and the mixture was refluxed for 1 h. After cooling, themixture was partitioned between water (10 mL) and EtOAc (10 mL). Theaqueous layer was further extracted with EtOAc (2×10 mL) and thecombined organic layers were washed with brine and dried over Na₂SO₄.After the removal of organic solvent, the crude product was purified bycolumn chromatography (EtOAc/Hexane: 1/2) to give 100 (25 mg, 41%) as awhite solid. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (d, 1H, J=1.6 Hz), 7.54 (d,1H, J=5.2 Hz), 7.44 (td, 1H, J=8.4, 1.6 Hz), 7.33-7.23 (m, 1H),7.16-7.06 (m, 2H), 7.04 (d, 1H, J=5.2 Hz), 5.28 (s, 2H), 4.23 (s, 3H);LC/MS: Method 1, retention time: 5.429 min; Method 2, retention time:3.819 min; HRMS: m/z (M+H⁺)=314.0761 (Calculated forC₁₆H₁₃FN₃OS=314.0763).

2,4-Methyl-6-[(2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(101). Analogue 101 was prepared in a similar procedure as analogue 81.Under N₂ atmosphere, to a sealed tube was added unsubstitutedpyridazinone (50 mg, 0.23 mmol), CuI (4.3 mg, 0.023 mmol),trans-cyclohexane-1,2-diamine (17.2 mg, 0.15 mmol), cesium carbonate(156 mg, 0.48 mmol), iodobenzene (51 μl, 0.46 mmol) and 1,4-dioxane. Thetube was sealed and the mixture was refluxed overnight. After cooling toroom temperature, the mixture was partitioned between water (5 mL) andDCM (5 mL). The aqueous layer was separated and further extracted withDCM (2×5 mL) and the combined organic layers were washed with brine anddried over Na₂SO₄. After the removal of organic solvent, the residue wasdirectly purified by preparative HPLC to give desired coupling product101 (17 mg, 25%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.30 (s,1H), 7.64-7.59 (m, 2H), 7.52-7.46 (m, 2H), 7.41-7.36 (m, 1H), 6.83 (q,1H, J=1.2 Hz), 4.29 (s, 3H), 2.66 (d, 3H, J=1.2 Hz); LC/MS: Method 1,retention time: 5.951 min; Method 2, retention time: 3.915 min; HRMS:m/z (M+H⁺)=296.0860 (Calculated for C₁₆H₁₄N₃OS=296.0858).

2,4-Methyl-6-pentyl-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone (102). ¹HNMR (400 MHz, CDCl₃) δ 8.23 (s, 1H), 6.81 (q, 1H, J=1.0 Hz), 5.30 (s,2H), 4.28 (t, 2H, J=5.6 Hz), 4.27 (s, 3H), 2.64 (d, 3H, J=1.0 Hz),1.90-1.80 (m, 2H), 1.42-1.35 (m, 4H), 0.90 (t, 3H, J=5.6 Hz); LC/MS:Method 1, retention time: 6.704; Method 2, retention time: 4.060 min;HRMS: m/z (MAI)=290.1326 (Calculated for C₁₅H₂₀N₃OS=290.1327).

2,4-Methyl-6-phenylmethyl-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(103). ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.44-7.40 (m, 2H),7.34-7.29 (m, 2H), 7.27-7.20 (m, 1H), 6.78 (q, 1H, J=1.0 Hz), 5.45 (s,2H), 4.26 (s, 3H), 2.63 (d, 3H, J=1.0 Hz,); LC/MS: Method 1, retentiontime: 6.254 min; Method 2, retention time: 3.992 min; HRMS: m/z(M+H⁺)=310.1011 (Calculated for C₁₇H₁₆N₃OS=310.1014).

2,4-Methyl-6-[3-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(104). ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.31-7.24 (m, 1H),7.21-7.17 (m, 1H), 7.10 (dt, 1H, J=10.0, 2.0 Hz), 6.94 (tdd, 1H, J=8.4,2.8, 0.8 Hz), 6.79 (q, 1H, J=1.2 Hz), 5.43 (s, 2H), 4.27 (s, 3H), 2.64(d, 3H, J=1.2 Hz); LC/MS: Method 1, retention time: 6.369 min; Method 2,retention time: 4.007 min; HRMS: m/z (M+H⁺)=328.0918 (Calculated forC₁₇H₁₅FN₃OS=328.0920).

2,4-Methyl-6-[(4-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(105). ¹H NMR (400 MHz, CDCl₃) δ 8.23 (s, 1H), 7.45-7.39 (m, 2H),7.02-6.97 (m, 2H), 6.80 (q, 1H, J=1.2 Hz), 5.42 (s, 2H), 4.26 (s, 3H),2.64 (d, 3H, J=1.2 Hz); LC/MS: Method 1, retention time: 6.346 min;Method 2, retention time: 4.000 min; HRMS: m/z (M+H⁺)=328.0919(Calculated for C₁₇H₁₅FN₃OS=328.0920).

2,4-Methyl-6-[(2-chlorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(106). ¹H NMR (400 MHz, CDCl₃) δ 8.23 (s, 1H), 7.39 (dd, 1H, J=7.2, 1.6Hz), 7.20-7.12 (m, 2H), 6.97 (ddd, 1H, J=6.8, 1.6, 0.8 Hz), 6.82 (q, 1H,J=1.2 Hz), 5.59 (s, 2H), 4.28 (s, 3H), 2.65 (d, 3H, J=1.2 Hz); LC/MS:Method 1, retention time: 6.646 min; Method 2, retention time: 4.064min; HRMS: m/z (M+H⁺)=344.0624 (Calculated for C₁₇H₁₅ClN₃OS=344.0624).

2,4-Methyl-6-[(3-chlorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(107). ¹H NMR (400 MHz, CDCl₃) δ 8.23 (s, 1H), 7.40-7.37 (m, 1H),7.31-7.27 (m, 1H), 7.27-7.22 (m, 2H), 6.80 (q, 1H, J=1.2 Hz), 5.42 (s,2H), 4.27 (s, 3H), 2.65 (d, 3H, J=1.2 Hz); LC/MS: Method 1, retentiontime: 6.704 min; Method 2, retention time: 4.081 min; HRMS: m/z(M+H⁺)=344.0623 (Calculated for C₁₇H₁₅ClN₃OS=344.0624).

2,4-Methyl-6-[(4-chlorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(108). ¹H NMR (400 MHz, CDCl₃) δ 8.18 (s, 1H), 7.37 (d, 2H, J=8.4 Hz),7.27 (d, 2H, J=8.4 Hz), 6.79 (q, 1H, J=1.2 Hz), 5.39 (s, 2H), 4.26 (s,3H), 2.63 (d, 3H, J=1.2 Hz); LC/MS: Method 1, retention time: 6.697 min;Method 2, retention time: 4.079 min; HRMS: m/z (M+H⁺)=344.0621(Calculated for C₁₇H₁₅ClN₃OS=344.0624).

2,4-Methyl-6-[(4-methylphenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(109). ¹H NMR (400 MHz, CDCl₃) δ 8.17 (s, 1H), 7.33 (d, 2H, J=8.0 Hz),7.12 (d, 2H, J=8.0 Hz), 6.78 (q, 1H, J=1.2 Hz), 5.40 (s, 2H), 4.26 (s,3H), 2.63 (d, 3H, J=1.2 Hz), 2.30 (s, 3H); LC/MS: Method 1, retentiontime: 6.563 min; Method 2, retention time: 4.044 min; HRMS: m/z(M+H⁺)=324.1170 (Calculated for C₁₈H₁₈N₃OS=324.1171).

2,4-Methyl-6-[(4-trifluoromethylphenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(110). ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.58 (d, 2H, J=8.4 Hz),7.51 (d, 2H, J=8.4 Hz), 6.79 (q, 1H, J=1.0 Hz), 5.48 (s, 2H), 4.26 (s,3H), 2.64 (d, 3H, J=1.0 Hz); LC/MS: Method 1, retention time: 6.819 min;Method 2, retention time: 4.082 min; HRMS: m/z (M+H⁺)=378.0886(Calculated for C₁₈H₁₅F₃N₃OS=378.0888).

2,4-Methyl-6-[(4-methoxyphenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(111). ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.39 (d, 2H, J=8.8 Hz),6.84 (d, 2H, J=8.8 Hz), 6.78 (q, 1H, J=1.0 Hz), 5.38 (s, 2H), 4.26 (s,3H), 3.77 (s, 3H), 2.63 (d, 3H, J=1.0 Hz); LC/MS: Method 1, retentiontime: 6.193 min; Method 2, retention time: 3.974 min; HRMS: m/z(M+H⁺)=340.1114 (Calculated for C₁₈H₁₈N₃O₂S=340.1120).

2,4-Methyl-6-[(2,4-difluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(112). ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.32-7.24 (m, 1H),6.85-6.76 (m, 3H), 5.47 (s, 2H), 4.27 (s, 3H), 2.64 (d, 3H, J=1.2 Hz);LC/MS: Method 1, retention time: 6.445 min; Method 2, retention time:4.012 min; HRMS: m/z (M+H⁺)=346.0825 (Calculated forC₁₇H₁₄F₂N₃OS=346.0826).

2,4-Methyl-6-[(2,6-difluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(113). ¹H NMR (400 MHz, CDCl₃) δ 8.13 (s, 1H), 7.28-7.20 (m, 1H),6.94-6.87 (m, 2H), 6.79 (q, 1H, J=1.2 Hz), 5.55 (s, 2H), 4.28 (s, 3H),2.63 (d, 3H, J=1.2 Hz); LC/MS: Method 1, retention time: 6.244 min;Method 2, retention time: 3.974 min; HRMS: m/z (M+H⁺)=346.0825(Calculated for C₁₇H₁₄F₂N₃OS=346.0826).

2,4-Methyl-6-[(2,3-difluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(114). ¹H NMR (CDCl₃, 400 MHz) δ 8.20 (s, 1H), 7.10-7.00 (m, 1H),7.01-6.97 (m, 2H), 6.80 (q, 1H, J=1.2 Hz), 5.54 (d, 2H, J=0.8 Hz), 4.28(s, 3H), 2.65 (d, 3H, J=1.2 Hz); LC/MS: Method 1, retention time: 6.443min; Method 2, retention time: 4.009 min; HRMS: m/z (M+H⁺)=346.0822(Calculated for C₁₇H₁₄F₂N₃OS=346.0826).

2,4-Methyl-6-[(2-chloro-6-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(115). ¹H NMR (CDCl₃, 400 MHz) δ 8.10 (s, 1H), 7.27-7.19 (m, 2H),7.04-6.99 (m, 1H), 6.80 (m, 1H), 5.62 (d, 2H, J=1.2 Hz), 4.29 (d, 3H,J=0.8 Hz), 2.63 (d, 3H, J=1.2 Hz); LC/MS: Method 1, retention time:6.504 min; Method 2, retention time: 4.040 min; HRMS: m/z(M+H⁺)=362.0528 (calculated for C₁₇H₁₄ClFN₃OS=362.0530).

2,4-Methyl-6-[(2,3,4-trifluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(116). ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.06-6.99 (m, 1H),6.92-6.84 (m, 1H), 6.80 (q, 1H, J=1.2 Hz), 5.48 (s, 2H), 4.27 (s, 3H),2.65 (d, 3H, J=1.2 Hz); LC/MS: Method 1, retention time: 6.617 min;Method 2, retention time: 4.044 min; HRMS: m/z (M+H⁺)=364.0727(Calculated for C₁₇H₁₃F₃N₃OS=364.0731).

2,4-Methyl-6-[(2,3,5,6-tetrafluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(117). ¹H NMR (400 MHz, CDCl₃) δ 8.14 (s, 1H), 7.07-6.88 (m, 1H), 6.80(q, 1H, J=1.2 Hz), 5.58 (t, 2H, J=1.2 Hz), 4.27 (s, 3H), 2.65 (d, 3H,J=1.2 Hz); LC/MS: Method 1, retention time: 6.557 min; Method 2,retention time: 4.034 min; HRMS: m/z (M+H⁺)=382.0637 (Calculated forC₁₇H₁₂F₄N₃OS=382.0637).

2,4-Methyl-6-[(3-methyl-2-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(118). ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.11-7.02 (m, 2H),6.96-6.92 (m, 1H), 6.80 (q, 1H, J=1.0 Hz), 5.52 (s, 2H), 4.28 (s, 3H),2.64 (d, 3H, J=1.0 Hz), 2.28 (s, 3H); LC/MS: Method 1, retention time:6.641 min; Method 2, retention time: 4.055 min; HRMS: m/z(M+H⁺)=342.1076 (Calculated for C₁₈H₁₇FN₃OS=342.1076).

2,4-Methyl-6-[(4-methyl-2,3-fluorophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(119). ¹H NMR (400 MHz, CDCl₃,) δ 8.19 (s, 1H), 6.92-6.80 (m, 2H), 6.80(q, 1H, J=1.2 Hz), 5.49 (s, 2H), 4.27 (s, 3H), 2.64 (d, 3H, J=1.2 Hz),2.56 (d, 3H, J=2.0 Hz); LC/MS: Method 1, retention time: 6.753 min;Method 2, retention time: 4.077 min; HRMS: m/z (M+H⁺)=360.0983(Calculated for C₁₈H₁₆F₂N₃OS=360.0982).

2,4-Methyl-6-[(2-fluoro-4-trifluoromethylphenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(120). ¹H NMR (400 MHz, CDCl₃) δ 8.21 (s, 1H), 7.36-7.32 (m, 3H), 6.81(q, 1H, J=1.2 Hz), 5.56 (s, 2H), 4.27 (s, 3H), 2.65 (d, 3H, J=1.2 Hz);LC/MS: Method 1, retention time: 6.920 min; Method 2, retention time:4.095 min; (TOFMS) m/z (M+H⁺)=396.0797 (Calculated forC₁₈H₁₄F₄N₃OS=396.0794).

2,4-Methyl-6-[(3-fluoro-4-methoxyphenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone(121). ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.21-7.16 (m, 1H),7.12-7.02 (m, 1H), 6.87-6.96 (m, 1H), 6.79 (q, 1H, J=1.2 Hz), 5.35 (s,2H), 4.26 (s, 3H), 3.85 (s, 3H), 2.65 (d, 3H, J=1.2 Hz); LC/MS: Method1, retention time: 6.620 min; Method 2, retention time: 3.982 min; HRMS:m/z (M+H⁺)=358.1023 (Calculated for C₁₈H₁₇FN₃O₂S=358.1026).

Example 2

This example illustrates additional embodiments of the compounds ofFormula Ia:

Compound 122: ¹H NMR (400 MHz, DMSO-d₆) 8 ppm 10.35 (s, 1H), 7.76 (d,J=8.80 Hz, 3H), 7.60 (d, J=8.80 Hz, 2H), 7.18-7.30 (m, 2H), 3.14-3.20(m, 4H), 2.91-3.01 (m, 4H), 2.08 (s, 3H).

Compound 123: ¹H NMR (400 MHz, DMSO-d₆) 8 ppm 7.75 (s, 1H), 7.19-7.37(m, 4H), 6.60 (d, J=8.80 Hz, 2H), 3.16 (d, J=4.50 Hz, 4H), 2.79-2.95 (m,4H); LC/MS: Method 1, retention time, 5.128 min; Method 2, retentiontime 3.748 min; HRMS: m/z (M+H⁺)=417.0634 (Calculated forC₁₆H₁₇N₃O₄S₂=417.0629).

Compound 124: ¹H NMR (400 MHz, DMSO-d₆) 8 ppm 7.18 (m, 3H), 6.98-7.09(m, 2H), 6.81-6.97 (m, 2H), 4.18-4.36 (m, 4H), 3.08-3.21 (m, 8H),1.66-1.83 (m, 2H); LC/MS: Method 1, retention time, 5.017 min; Method 2,retention time 3.704 min; HRMS: m/z (M+H⁺)=453.1035 (Calculated forC₁₉H₂₃N₃O₆S₂=453.1028).

Compound 125: ¹H NMR (400 MHz, DMSO-d₆) 8 ppm 7.30-7.40 (m, 2H),7.13-7.23 (m, 2H), 6.95-7.09 (m, 1H), 6.52-6.65 (m, 2H), 4.19-4.36 (m,4H), 3.20-3.28 (m, 2H), 3.15 (m, 4H), 3.02-3.12 (m, 2H), 1.62-1.79 (m,2H). Method 1, retention time, 5.100 min; Method 2, retention time 3.741min; HRMS: m/z (M+H⁺)=453.1036 (Calculated for C₁₇H₁₅FN₃OS=453.1028).

Compound 126: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.16-10.29 (m, 1H),7.99-8.15 (m, 1H), 7.76 (dd, J=8.12, 0.88 Hz, 1H), 7.50 (t, J=8.02 Hz,1H), 7.39 (d, J=8.02 Hz, 1H), 6.81-6.99 (m, 2H), 3.75 (s, 3H), 3.35-3.44(m, 2H), 3.30 (m, 4H), 3.23 (t, J=5.77 Hz, 2H), 2.04 (s, 3H), 1.69-1.89(m, 2H); LC/MS: Method 1, retention time, 5.349 min; Method 2, retentiontime 3.907 min; HRMS: m/z (M+H⁺)=503.1004 (Calculated forC₂₀H₂₃N₃O₆S₂=503.0996).

Compound 127: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.31 (s, 1H), 7.71 (m,4H), 6.76-7.00 (m, 2H), 3.82 (s, 3H), 3.16-3.32 (m, 8H), 2.05 (s, 3H),1.78 (m, 2H).

Compound 128: ¹H NMR (400 MHz, DMSO-d₆) 5 ppm 7.16-7.30 (m, 2H),6.99-7.10 (m, 1H), 6.85-6.96 (m, 2H), 4.29 (q, J=5.09 Hz, 4H), 3.82 (s,3H), 3.30-3.49 (m, 4H), 3.14-3.25 (m, 4H), 1.75 (m, 2H). LC/MS: Method1, retention time, 5.897 min; Method 2, retention time 3.782 min; HRMS:m/z (M+H⁺)=504.0851 (Calculated for C₂₀H₂₂N₂O₇F₂S₂=504.0836).

Compound 129: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.17-10.35 (s, 1H),7.92-8.09 (m, 1H), 7.71-7.85 (m, 1H), 7.44-7.57 (m, 1H), 7.24-7.39 (m,1H), 6.95-7.17 (m, 3H), 4.15-4.41 (m, 4H), 2.82-3.03 (m, 8H), 2.04 (s,3H).

Compound 130: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.35 (s, 1H), 7.76 (d,J=8.80 Hz, 2H), 7.58 (d, J=8.80 Hz, 2H), 7.08 (dd, J=4.30, 2.35 Hz, 2H),6.98 (d, J=9.00 Hz, 1H), 4.29 (d, J=3.72 Hz, 4H), 2.91 (m, 8H), 2.07 (s,3H).

Compound 131: ¹H NMR (400 MHz, DMSO-d₆) 5 ppm 10.24 (s, 1H), 7.98-8.13(m, 1H), 7.72-7.79 (m, 1H), 7.49 (s, 1H), 7.34-7.40 (m, 1H), 7.18 (m,2H), 6.96-7.03 (m, 1H), 4.20-4.35 (m, 4H), 3.37-3.45 (m, 4H), 3.09-3.22(m, 4H), 2.04 (s, 3H), 1.70-1.82 (m, 2H); LC/MS: Method 1, retentiontime, 5.131 min; Method 2, retention time 3.744 min; HRMS: m/z(M+H⁺)=495.1133 (Calculated for C₂₁H₂₅N₃O₇S₂=495.1134).

Compound 132: ¹H NMR (400 MHz, DMSO-d₆) 8 ppm 10.25-10.36 (m, 1H), 7.73(m, 2H), 7.67 (m, 2H), 7.17 (m, 2H), 7.01 (m, 1H), 4.28 (d, J=4.11 Hz,4H), 3.16 (m, 8H), 2.05 (s, 3H), 1.74 (m, 2H).

Compound 133: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.27 (s, 1H), 7.94-8.08(m, 1H), 7.73-7.88 (m, 1H), 7.51 (t, J=8.02 Hz, 1H), 7.32 (d, J=8.02 Hz,1H), 6.89 (d, J=11.54 Hz, 2H), 3.84 (s, 3H), 2.88-3.21 (m, 8H), 2.04 (s,3H); LC/MS: Method 1, retention time, 5.296 min; Method 2, retentiontime 3.773 min; HRMS: m/z (M+H⁺)=489.0845 (Calculated forC₁₉H₂₁N₃O₆F₂S₂=489.0840).

Example 3

This example illustrates some of the properties of the compounds of theinvention.

Reagents: Kinase-Glo was obtained from Promega (Madison, Wis.). ATP,PEP, LDH and NADH were from Sigma. Reagents and solvents were purchasedfrom Sigma, Alfa Aesar, Acros, Enamine, Oakwood Products, MatrixScientific or Chem-Impex International.

Luminescent pyruvate kinase-luciferase coupled assay. Production of aluminescent signal based on the generation of ATP by pyruvate kinase wasdetermined by using the ATP-dependent enzyme firefly luciferase. ThreeμL of substrate mix (at r.t.) in assay buffer (50 mM imidazole pH 7.2,50 mM KCl, 7 mM MgCl₂, 0.01% tween 20, 0.05% BSA) was dispensed intoKalypsys white solid bottom 1,536 well microtiter plates using abottle-valve solenoid-based dispenser (Kalypsys). The finalconcentrations of substrates in the assay were 0.1 mM ADP and 0.5 mMPEP. Twenty-three nL of compound were delivered with a 1,536-pin arraytool and 1 μl of enzyme mix in assay buffer (final concentration, 0.1 nMpyruvate kinase, 50 mM imidazole pH 7.2, 0.05% BSA, 4° C.) was added.Microtiter plates were incubated at r.t. for 1 hour and 2 uL ofluciferase detection mix (Kinase-Glo, Promega at 4° C. protected fromlight) was added and luminescence was read with a ViewLux (Perkin Elmer)using a 5 second exposure/plate. Data was normalized for AC50 values tocontrol columns containing uninhibited enzyme (n), and AC₁₀₀ inhibition(i) according the following equation: Activation (%)=[(c−n)/(n−i)]*100where c=compound, n=DMSO neutral, i=no enzyme control. A % activity of100% is approximately a 2-fold increase over basel assay signal (%Activation by FBP was variable but averaged 100%). Monitoring ofactivation was accomplished using enzyme at 3× the final concentration.The primary qHTS data and confirmatory data are available in PubChem(AIDS: 1631, 1634, and 1751). Follow-up of synthesized analogs wasdetermined using the same protocol with the exception that the enzymeconcentrations for isoforms PKM1, L and R were 1 nM, 0.1 nM, and 0.1 nMrespectively (PubChem AIDs for M1, L and R bioluminescent assays are1780, 1781, and 1782).

Fluorescent pyruvate kinase-lactate dehydrogenase coupled secondaryassay. All compounds were also tested in a kinetic mode by coupling thegeneration of pyruvate by pyruvate kinase to the depletion of NADHthrough lactate dehydrogenase. For PKM2, 3 μL of substrate mix (finalconcentration, 50 mM Tris-Cl pH 8.0, 200 mM KCl, 15 mM MgCl₂, 0.1 mMPEP, 4.0 mM ADP, and 0.2 mM NADH) was dispensed into Kalypsysblack-solid 1,536 well plates using the Aurora Discovery BioRAPTR FlyingReagent Dispenser (FRD; Beckton-Dickenson, Franklin Lakes, N.J.) and 23nL of compounds were delivered a Kalypsys pin tool and then 1 μL ofenzyme mix (final concentrations, 10 nM hPK-M2 and 1 μM of LDH) wasadded. Plates were immediately placed in ViewLux (Perkin Elmer) and NADHfluorescence was determined at 30 second exposure intervals for between3 and 6 minutes. Data were normalized to the uninhibited and EC₁₀₀activation using known activators such as fructose-1,6-bis-phosphate.The data has been deposited in PubChem (AID: 1540). Follow-up ofsynthesized analogs was determined using the same protocol (PubChem AIDsfor L, M1 and R bioluminescent assays are 1541, 1542, and 1543). Thisassay was also used to determine the K_(M)'s for PEP and ADP in thepresence and absence of activator. Conversion of fluorescent units topmols of NADH was performed using a standard curve of known NADHconcentrations. Data was collected on the Perkin Elmer Viewlux.

Mode of action. The mode of action was examined for each of these leadchemotypes through analysis of the activators on the kinetics of PEP andADP utilization by the enzyme. As discussed, FBP is known toallosterically activate PKM2 through induction of an enzyme state with ahigh affinity for PEP. In the absence of activator, hPK shows lowaffinity for PEP (K_(M) ˜1.5 mM). In the presence of 1 or FBP the K_(M)for PEP decreased to 0.26±0.08 mM or 0.1±0.02 mM, respectively.Comparison of the ADP titration in the presence and absence ofactivators shows that these kinetics are not significantly affected(K_(M) for ADP ˜0.1 mM in either condition; V_(max) values within 20% ofeach other). Thus, the primary lead NCGC00030335 (the substitutedN,N′-diarylsulfonamide 1) increased the affinity of PKM2 for PEP (FIG.1A) while having less affect on ADP kinetics (FIG. 1B).

Identification of NCGC00030355 (1): Following the qHTS the CRC data wassubjected to a classification scheme to rank the quality of the CRCs asdescribed by Inglese and co-workers (Proc. Natl. Acad. Sci. USA 2006,103, 11473-11478) (see FIG. 3). Agents, including NCGC00030335 (1), werechosen for follow-up based upon their curve class ranking. Briefly, CRCsare placed into four classes. Class 1 contains complete CRCs showingboth upper and lower asymptotes and r² values >0.9. Class 2 containsincomplete CRCs lacking the lower asymptote and shows r² values greaterthan 0.9. Class 3 curves are of the lowest confidence because they aredefined by a single concentration point where the minimal acceptableactivity is set at 3 SD of the mean activity calculated from the lowesttested concentration. Finally, class 4 contains compounds that do notshow any CRCs and are therefore classified as inactive.

FIG. 3 shows an example qHTS data and classification scheme forassignment of resulting curve-fit data into classes. Top, qHTS curve-fitdata from AID 361 binned into curve classifications 1-4 basedclassification criteria. Below, Examples of curves fitting the followingclassification criteria: Class 1 curves display two asymptotes, aninflection point, and r2≧0.9; subclasses 1a (blue) vs. 1b (orange) aredifferentiated by full (>80%) vs. partial (≦80%) response. Class 2curves display a single left-hand asymptote and inflection point;subclasses 2a (blue) and 2b (orange) are differentiated by a maxresponse and r2, >80% and >0.9 or <80% and <0.9, respectively. Class 3curves have a single left-hand asymptote, no inflection point, and aresponse >3SD the mean activity of the sample field. Class 4 definesthose samples showing no activity across the concentration range.

SAR of substituted N,N′-diarylsulfonamides and selected analogues. Thelead structure 1 identified from the primary screen was found to possessAC₅₀ values versus PKM2 of 0.063±0.02 μM and 0.111±0.03 μM and maximumresponses versus PKM2 (relative to activation by FBP) of 122.1% and92.2%, respectively (Table 1). In the LDH assay, that used highsaturating ADP levels and low (0.1 mM) levels of PEP, average greaterefficacy but lower potency was found for compound 1 showing AC₅₀ valueof 0.3±0.1 μM but with maximum response of 224%. The initial focusinvolved symmetric versions of the N,N′-diarylsulfonamides. As such,symmetry was examined utilizing the 6-(2,3-dihydrobenzo[b][1,4]dioxine)heterocycle (analogue 2) and the 4-methoxybenzene ring (analogue 3).Each analogue had slightly diminished AC₅₀ values (270 nM and 171 nM,respectively). From here, one aryl sulfonamide unit was held constantwhile exploring the SAR of the other aryl sulfonamide. Compounds 6-18are representative examples from this strategy whereby the6-(2,3-dihydrobenzo[b][1,4]dioxine) heterocycle remained constant andthe 4-methoxybenzene ring was changed utilizing standard phenyl ringanalogues. While there were selective tendencies associated withelectron withdrawing and electron donating substituents, as a wholethere was no discernable trend associated with either strategy.Substitutions of small and modest size were accepted at the ortho, metaand para positions. Moderate to large substitutions, however, were nottolerated at the para position. This is demonstrated by comparison ofanalogues 11 and 12 in which replacing the para-methoxy substituent in11 with the para-n-propyl group in 12 shows diminished activity (thisgeneral trend was seen with numerous analogues; data not shown). Themost effective substitutions involved electron withdrawing groups in the2- and 6-positions of the phenyl ring [for instance 2,6-difluorobenzene(analogue 9, AC₅₀=65±25 nM, maximum response=94.4%),2,6-difluoro-4-methoxybenzene (analogue 11, AC₅₀=28±9 nM, maximumresponse=91.8%) and 2,6-difluoro-3-phenol (analogue 13, AC₅₀=52±14 nM,maximum response=95.3%)]. In an attempt to place additional electrondensity in the ortho-position of this phenyl ring a pyridine analoguewas synthesized placing the nitrogen at the 2-position of the aromaticring (analogue 18) and additionally oxidized to the N-oxide 19. Thisdesign was not successful as 19 displayed both reduced potency andmaximum response (AC₅₀>10 μM in both assays). Given the establishedadvantage of the di-fluoro analogues, we next chose to hold the2,6-difluorobenzene ring constant and vary the opposite side with bothsubstituted phenyl rings and various heterocycles. Numerous analogues ofthis class were synthesized and tested and compounds 21-29 are goodrepresentations of these analogues' SAR. The various substituted phenylrings generally resulted in active compounds as represented by 21(AC₅₀=90±16 nM, maximum response=102.0%) and 22 (AC₅₀=66±nM, maximumresponse=74.3%). Both compound 21 and 22 showed comparable activity inthe LDH coupled assay with AC₅₀'s=211±50 nM (maximum response=124±30)and 172±29 nM (maximum response=85±8%), respectively. However, noneprovided significant improvements in potency or maximum response.Altering the heterocycle from the 6-(2,3-dihydrobenzo[b][1,4]dioxine)moiety had varying consequences. Several heterocycles were toleratedincluding the 7-(3,4-dihydro-2H-benzo[b][1,4]dioxepine) moiety (analogue23, AC₅₀=103±30 nM, maximum response=100.4%) and the6-(2-methylbenzo[d]thiazole) moiety (analogue 29, AC₅₀=86±6 nM, maximumresponse=103.6%). As well, in the LDH assay 23 and 29 showed potenciesof ˜0.5 μM with maximum responses of 109±11% and 156±11%. The 2-napthyland 6-(2,2-dimethylchroman) derivatives provided significant enhancementin terms of maximum response (analogues 26 and 27, AC₅₀=66±4 nM and93±12 nM, maximum response=138.0% and 119.3%, respectively). Compound 26also showed good response in the LDH assay with an AC₅₀=220±53 nM and amaximum response of 161±29%. The sulfone derivatives that were exploredshowed loss of potency. Interestingly, this loss was more severe whenthe sulfone moiety bridged the piperidine ring system to the6-(2,3-dihydrobenzo[b][1,4]dioxine) system relative to substitutedphenyl rings (i.e. 2,6-difluorophenyl) as illustrated by analogues 19and 30 (AC₅₀=254±47 nM and 863±56 nM, maximum response=104.3% and110.0%, respectively, similar values were also observed in the LDHassay).

TABLE 1 (Ia)

Compound hPK, M2 hPK, M2 No. X R¹ R² AC₅₀(μM)^(a) Max. Res.^(b)  1 N4-methoxyphenyl 6-(2,3-dihydro-benzo[b][1,4] 0.111 ± 0.03 92.2 ± 12.0dioxinyl)  2 N 6-(2,3-dihydro- 6-(2,3-dihydro-benzo[b][1,4] 0.270 ± 0.0889.7 ± 2.2  benzo[b][1,4]dioxinyl) dioxinyl)  3 N 4-methoxyphenyl4-methoxyphenyl 0.171 ± 0.01 87.6 ± 16.2  4 N 4-cyanophenyl6-(2,3-dihydro-benzo[b][1,4] 0.029 ± 0.02 44.1 ± 6.1  dioxinyl)  5 N4-chlorophenyl 6-(2,3-dihydro-benzo[b][1,4] 0.154 ± 0.08 99.6 ± 2.5 dioxinyl)  6 N 4-fluorophenyl 6-(2,3-dihydro-benzo[b][1,4] 0.094 ± 0.0399.7 ± 54.2 dioxinyl)  7 N 3-fluorophenyl 6-(2,3-dihydro-benzo[b][1,4]0.316 ± 0   106.7 ± 8.8  dioxinyl)  8 N 2-fluorophenyl6-(2,3-dihydro-benzo[b][1,4] 0.089 ± 0.03 114.4 ± 4.0  dioxinyl)  9 N2,6-difluorophenyl 6-(2,3-dihydro-benzo[b][1,4] 0.065 ± 0.03 94.4 ± 2.8 dioxinyl) 10 N 2,4,5-trifluorophenyl 6-(2,3-dihydro-benzo[b][1,4] 0.090± 0.01 104.9 ± 7.6  dioxinyl) 11 N 2,6-difluoro-4-6-(2,3-dihydro-benzo[b][1,4] 0.028 ± 0.01 91.8 ± 9.6  methoxyphenyldioxinyl) 12 N 2,5-difluoro-3- 6-(2,3-dihydro-benzo[b][1,4] 0.757 ± 0.2269.2 ± 10.4 propylphenyl dioxinyl) 13 N 2,6-difluoro-3-6-(2,3-dihydro-benzo[b][1,4] 0.052 ± 0.01 95.3 ± 8.4  hydroxyphenyldioxinyl) 14 N 2,4-difluorophenyl 6-(2,3-dihydro-benzo[b][1,4] 0.124 ±0.03 112.9 ± 6.3  dioxinyl) 15 N phenyl 6-(2,3-dihydro-benzo[b][1,4]0.202 ± 0.04 108.2 ± 4.3  dioxinyl) 16 N 3- 6-(2,3-dihydro-benzo[b][1,4]0.209 ± 0.07 39.3 ± 6.2  (trifluoromethyl)phenyl dioxinyl) 17 N3-methoxyphenyl 6-(2,3-dihydro-benzo[b][1,4] 0.113 ± 0.04 90.0 ± 4.5 dioxinyl) 18 N 2-pyridyl 6-(2,3-dihydro-benzo[b][1,4] 0.542 ± 0.04 103.1± 6.6  dioxinyl) 19 N 2-pyridyl-1-oxide 6-(2,3-dihydro-benzo[b][1,4] >1081.5 ± 3.2  dioxinyl) 20 CH 2,6-difluorophenyl6-(2,3-dihydro-benzo[b][1,4] 0.254 ± 0.05 104.3 ± 5.1  dioxinyl) 21 N4-methoxyphenyl 6-(2,3-dihydro-benzo[b][1,4] 0.090 ± 0.02 102.0 ± 9.2 dioxinyl) 22 N 2,6-difluorophenyl 2,6-difluorophenyl 0.066 ± 0.01 74.3 ±9.8  23 N 2,6-difluorophenyl 7-(3,4-dihydro-2H- 0.103 ± 0.03 100.4 ±6.6  benzo[b][1,4]dioxepine) 24 N 2,6-difluorophenyl5-benzo[b][1,3]dioxinyl 0.191 ± 0.06 61.0 ± 1.2  25 N 2,6-difluorophenyl7-(4-methyl-3,4-dihydro-2H-  2.71 ± 0.18 93.5 ± 5.9 pyrido[3,2-b][1,4]oxazine) 26 N 2,6-difluorophenyl 2-naphthalenyl 0.066± 0   138.0 ± 11.3  27 N 2,6-difluorophenyl 6-(2,2-dimethylchroman)yl0.093 ± 0.01 119.3 ± 5.4  28 N 2,6-difluorophenyl5-(1-methyl-1H-indolyl) 0.387 ± 0.07 91.1 ± 4.7  29 N 2,6-difluorophenyl6-(2-methylbenzo[d]thiazolyl) 0.086 ± 0.01 103.6 ± 5.8  30 CH2,6-difluorophenyl 6-(2,3- 0.863 ± 0.12 110.0 ± 5.4 dihydrobenzo[b][1,4]dioxinyl) 31 N 2,6-difluorophenyl 6-(2,3- 0.866 ±0.15 119.9 ± 7.3  dihydrobenzo[b][1,4]dioxinyl) ^(a)AC50 values weredetermined utilizing the luminescent pyruvate kinase-luciferase coupledassay and the data represents the results from three separateexperiments. ^(b)Max Res. (Maximum Response) is % activity thatrepresents % activation at 57 μM of compound.

Additional compounds of Formula Ia and their properties are set forth inTable 2.

TABLE 2 KinaseGlo LDH Compound hPK, M2 hPK, M2 hPK, M2 hPK, M2 No. X R¹R² AC₅₀(μM) Max. Res.^(a) AC₅₀(μM) Max. Res.^(a) 122 N2,6-difluorophenyl p-acetylaminophenyl 123 N 2,6-difluorophenylp-aminophenyl 0.6506 82.61 0.4105 170.98 *124 N m-aminophenyl6-(2,3-dihydro- 0.0326 88.94 0.0919 169.71 benzo[b][1,4] dioxinyl) *125N p-aminophenyl 6-(2,3-dihydro- 0.1031 86.64 0.1634 131.59 benzo[b][1,4]dioxinyl) *126 N m- 2,6-difluoro-4- 2.9063 83.79 2.9063 114.61acetylaminophenyl methoxyphenyl *127 N p- 2,6-difluoro-4-acetylaminophenyl methoxyphenyl *128 N 2,6-difluoro-4- 6-(2,3-dihydro-0.0366 96.67 0.058 159.85 methoxyphenyl benzo[b][1,4] dioxinyl) 129 N m-6-(2,3-dihydro- acetylaminophenyl benzo[b][1,4] dioxinyl) 130 N p-6-(2,3-dihydro- acetylaminophenyl benzo[b][1,4] dioxinyl) *131 N m-6-(2,3-dihydro- 0.8191 91.11 0.8191 123.67 acetylaminophenylbenzo[b][1,4] dioxinyl) *132 N p- 6-(2,3-dihydro- 4.1053 81.07 2.5902123.97 acetylaminophenyl benzo[b][1,4] dioxinyl) 133 N m-2,6-difluoro-4- 1.63 60.00 1.0312 94.88 acetylaminophenyl methoxyphenyl134 N m-aminophenyl 2,6-difluoro-4- 0.0231 87.00 0.073 141.67methoxyphenyl 135 N m-aminophenyl 6-(2,3-dihydro- 0.0411 82.08 0.145798.13 benzo[b][1,4] dioxinyl) 136 N m- 6-(2,3-dihydro- 0.081 63.00 nd nd(ethylamino)- benzo[b][1,4] phenyl dioxinyl) 137 N m-aminophenyl2,6-difluorophenyl 0.0919 81.51 0.2058 154.42 138 N m-(N,N-6-(2,3-dihydro- 0.092 54.00 nd nd dimethylamino)- benzo[b][1,4] phenyldioxinyl) 139 N p-aminophenyl 2,6-difluoro-4- 0.1298 92.46 0.1834 166.00methoxyphenyl 140 N m-hydroxyphenyl 2,6-difluorophenyl 0.1834 89.610.1834 148.41 *141 N m-aminophenyl 2,6-difluoro-4- 0.2058 93.20 0.1457157.96 methoxyphenyl 142 N m- 6-(2,3-dihydro- 0.29 67.00 nd nd(methylamino)- benzo[b][1,4] phenyl dioxinyl) *143 N p-aminophenyl2,6-difluoro-4- 1.2982 89.36 1.2982 150.25 methoxyphenyl 144 N2,6-difluoro-4- 2-thiophenyl 0.0366 95.08 0.0517 125.28 methoxyphenyl145 N 2,6-difluoro-4- 2-furanyl 0.1834 82.49 0.1157 106.13 methoxyphenyl146 N 2-amino-4-pyridyl 6-(2,3-dihydro- 1.6 81.00 nd nd benzo[b][1,4]dioxinyl) 151 N p-aminophenyl 6-(2,3-dihydro- 0.4105 91.84 0.3659 174.46benzo[b][1,4] dioxinyl) *n = 2; others n = 1; R³—R¹⁰═H. ^(a)Max Res.(Maximum Response) is % activity that represents % activation at 57 μMof compound.

Several linear diamines and several alternate diamine core ring systemswere examined. For these studies we retained the 2,6-difluorophenyl and6-(2,3-dihydrobenzo[b][1,4]dioxine) heterocycle as the two arylsubstituents to afford comparative uniformity. The results are shown inTable 1 and demonstrate that the piperazine and the related1,4-diazepane (analogue 31, AC₅₀=866 nM, maximum response=119.9%) hadclear advantages over other diamine moieties. Ligations with lineardiamines ranging from 2- to 6-carbons in length (analogues 32-36) werefound to have diminished potencies, as shown in Table 3. Cis and transversions of the cyclohexane-1,4-diamine ligation conferred similar lossin activation potency. Interestingly, the trans version of this analogueperformed significantly better than the cis version (analogues 37 and38, AC₅₀=2.11±0.64 μM and 37.1±5 μM, maximum response of 90.8% and60.7%, respectively; AC₅₀s of 7±1.5 μM and 56±11% for both compounds inthe LDH assay). Numerous analogues with one secondary amine contained in4-, 5- and 6-membered rings and one exocyclic primary amine wereexamined (analogues 39-44) and found to be less active than the originallead compounds in both assays. In addition to the derivatives shown inTable 3, numerous bicyclic and spirocyclic diamines were examined (forinstance 2,6-diazabicyclo[3.2.2]nonane and 2,7-diazaspiro[4.4]nonane)and found to be less active that the corresponding piperazine and1,4-diazepane analogues (data not shown).

TABLE 3

Compound hPK, M2 hPK, M2 No. L AC₅₀(μM)^(a) Max. Res.^(b) 32N,N′-(ethane-1,2-diyl) >15 60.3 ± 20.6 33 N,N′-(propane-1,3-diyl) 3.85 ±0.53 105.7 ± 5.1  34 N,N′-(butane-1,4-diyl) 7.97 ± 4.05 113.0 ± 14.6  35N,N′-(pentane-1,5-diyl) 2.33 ± 0.16 113.9 ± 1.4  36N,N′-(hexane-1,6-diyl) 4.83 ± 0.31 110.4 ± 3.0  37 N,N′-((trans)- 2.11 ±0.48 90.8 ± 12.4 cyclohexane-1,4-diyl) 38 N,N′-((cis)-cyclohexane- >3560.7 ± 5.6  1,4-diyl) 39

3.69 ± 1.26 100.9 ± 1.9  40

9.00 ± 4.5  99.6 ± 3.1  41

> 15 82.4 ± 18   42

>10 83.7 ± 24.2 43

4.47 ± 0   93.3 ± 9   44

3.05 ± 0.2  108.3 ± 5.3  ^(a)AC50 values were determined utilizing theluminescent pyruvate kinase-luciferase coupled assay and the datarepresents the results from three separate experiments. ^(b)Max Res.(Maximum Response) is % activity that represents % activation at 57 μMof compound.

Further investigations were made into substitutions directly on thepiperazine ring. To this end, several piperazine rings were synthesizedand evaluated with a single methyl addition proximal to either the2,6-difluorophenyl or 6-(2,3-dihydrobenzo[b][1,4]dioxine) heterocycle.Additional consideration was given to the absolute stereochemistry ofthe methyl group. The results are detailed in Table 43 and show thatthese analogues were less potent than the unmodified ring systems.Another piperazine ring modification was the incorporation of a carbonylmoiety alpha to the ring nitrogens. Here, the amine to lactam conversionproximal to the 6-(2,3-dihydrobenzo[b][1,4]dioxine) heterocycle resultedin an active derivative (analogue 49, AC₅₀=114±10 nM, maximumresponse=105.1%; LDH assay showed AC₅₀=0.44±0.24 μM, maximumresponse=87±37%). The same amine to lactam conversion adjacent to the2,6-difluorobenzene resulted in a loss of potency (analogue 50,AC₅₀=2.42±0.94 μM, maximum response=96.9%; LDH assay showed AC₅₀=3.16μM, maximum response=82%). The activities of these agents againdemonstrate the lack of symmetric SAR for this chemotype.

TABLE 4 Compound hPK, M2 hPK, M2 No. AC₅₀(μM)^(a) Max. Res.^(b) 45 4.34± 0.74 109.0 ± 9.4 46 3.10 ± 1.17  98.8 ± 2.6 47 9.18 ± 2.56 107.6 ± 9.948 2.96 ± 0.2  107.9 ± 8.4 49 0.114 ± 0.02  105.1 ± 9  50 2.42 ± 0.16 96.9 ± 5.6 ^(a)AC50 values were determined utilizing the luminescentpyruvate kinase-luciferase coupled assay and the data represents theresults from three separate experiments. ^(b)Max Res. (Maximum Response)is % activity that represents % activation at 57 μM of compound.

Selectivity of chosen N,N′-diarylsulfonamide analogues. With a betterunderstanding of the SAR for this chemotype, we next concerned ourselveswith the selective activation of PKM2 versus PKM1, PKR and PKL. Anappropriate tool compound aimed at further delineating the role of PKM2as a critical contributor in the Warburg effect requires a high degreeof selective activation of PKM2 relative to other PK targets withparticular consideration for PKM1. Members of each chemotype wereassayed versus PKM1, PKR and PKL. All analogues in theN,N′-diarylsulfonamides class were found to be inactive versus PKM1.This is consistent with the lack of allosteric regulation for the PKM1isoform. Data varied from compound to compound, however all thecompounds showed weak or no response versus PKR (<32% in both assayformats) and similar selectivity was observed for PKL (maximum response<30%, both assay formats). The selectivity for NCGC00030335 (1) is shownin FIG. 2; PKM2 (open circles), PKM1 (filled squares), PKL (opensquares) and PKR (filled circles).

SAR of substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinones and selectedanalogues. As a standard practice, the lead substitutedthieno[3,2-b]pyrrole[3,2-d]pyridazinone NCGC00031955 (66) wasre-synthesized and found to possess an AC₅₀ value of 63±20 nM andmaximum response of 122.1% in the luciferase-coupled assay and alsoshowed good potency and efficacy in the LDH coupled reaction (AC₅₀ valueof 326±90 nM, maximum response of 224±64%). In general, this seriesshowed stronger activation than the analogs of 1. The SAR from theluciferase-coupled assay and mention the LDH-coupled assay for specificexamples but the entire dataset for both assays is available in PubChem.Our first SAR evaluations involved changes directly to the heterocycliccore structure while retaining the standard 2-fluorobenzyl substitutionfrom the pyridazinone ring amide (Table 4). Steric expansions of themethyl group at the 2 position of the thiophene ring were typically welltolerated [for instance the ethyl and isopropyl analogues 68(AC₅₀=100±33 nM, maximum response=105.3% and 69 (AC₅₀=142±16 nM, maximumresponse=100%)]. Compound 69 did show weaker potency in the LDH assay(1.8±16 μM) but maintained an impressive max response (302%). Ingeneral, comparable potencies for these compounds were observed in theLDH assay, yet the efficacies were typically 2-3 fold higher. Removal ofthe methyl group resulted in a loss of potency and efficacy [see 70(AC₅₀=605 nM, maximum response=93.2)]. Insertions of heteroatomstypically resulted in improved potency including SMe [see 7 (AC₅₀=24±8nM, maximum response=96.3%; LDH assay showed an AC₅₀=110±10 nM andmaximum response=259%)] and S(O)Me [see 73 (AC₅₀=25±6 nM, maximumresponse=97.9%; LDH assay showed an AC₅₀=190±10 nM and maximumresponse=211%)]. Interestingly, oxidation past the sulfoxide to thesulfone resulted in a completely inactive analogue. Carbonyls andalcohols were examined and found to retain good potencies and maximumresponses [for instance 84 (AC₅₀=16±6 nM, maximum response=99.8%; LDHassay, AC₅₀=100±10 nM and maximum response=239%), 85 (AC₅₀=48±14 nM,maximum response=103.4%; LDH assay, AC₅₀=220±30 nM and maximumresponse=155%) and 87 (AC₅₀=11±3 nM, maximum response=107.6%; LDH assay,AC₅₀=150±30 nM and maximum response=200%)]. In stark contrast tosubstitutions on the 2 position of the thiophene ring, the methyl groupon the pyrrole ring nitrogen was found to be an absolute necessity.Alterations from the methyl to the ethyl and isopropyl groups wereineffective and lack of substitution was found to result in an inactiveanalogue as well. Further, amides and sulfonamides were examined at thismoiety and were not tolerated (data not shown). Addition of a methylgroup to the 6 position of the pyridazinone ring was also not allowed[see 92 (AC₅₀>30 μM, maximum response <80%, in both assays)]. Alterationfrom the pyridazinone to a pyrimidinone ring system was additionallyproblematic [see 100 (AC₅₀>35 μM, maximum response <80%, in bothassays)]. The necessity of the benzyl substituent was proven throughexamination of the corresponding phenyl analogue 101 and the n-pentylanalogue 102, both of which had marked loss of potency.

TABLE 5 (II)

Compound hPK, M2 hPK, M2 No. R¹¹ R¹² R¹³ AC₅₀ (μM)^(a) Max. Res.^(b) 66Me Me 2-fluoro 0.063 ± 0.02 122.1 ± 6.1 68 Et Me 2-fluoro 0.100 ± 0.03105.3 ± 5.8 69 iPr Me 2-fluoro 0.142 ± 0.02 105.8 ± 6.2 70 H Me 2-fluoro0.605 ± 0.18  93.2 ± 6.6 71 OMe Me 2-fluoro 0.086 ± 0.04 107.0 ± 8.7 72SMe Me 2-fluoro 0.024 ± 0.01  96.3 ± 3.8 73 S(O)Me Me 2-fluoro 0.025 ±0.01  97.9 ± 3.1 80 NO₂ Me 2-fluoro 0.018 ± 0.01 113.0 ± 3.5 81 NHA_(c)Me 2-fluoro >25  58.6 ± 23.5 82 CN Me 2-fluoro 0.047 ± 0.02  84.1 ± 5.583 COOMe Me 2-fluoro 0.084 ± 0.03  70.4 ± 12.9 84 CHO Me 2-fluoro 0.016± 0.01  99.8 ± 6.5 85 CH₂OH Me 2-fluoro 0.048 ± 0.01 103.4 ± 7.2 86B(OH)₂ Me 2-fluoro >10 101.3 ± 1.4 87 COMe Me 2-fluoro 0.011 ± 0   107.6± 4.9 88 CHOH(Me) Me 2-fluoro 0.136 ± 0.01 119.7 ± 2.6 89 Me H 2-fluoroNA  32.9 ± 3.8 90 Me Me 2-fluoro  5.9 ± 1.7  95.6 ± 6.3 ^(a)AC50 valueswere determined utilizing the luminescent pyruvate kinase-luciferasecoupled assay and the data represents the results from three separateexperiments. ^(b)Max Res. (Maximum Response) is % activity thatrepresents % activation at 57 μM of compound. See Methods fornormalization.

TABLE 6 (II)

Compound hPK, M2 hPK, M2 No. R¹¹ AC₅₀ (μM)^(a) Max. Res.^(b)  662-fluoro 0.063 ± 0.02 122.1 ± 6.1 103 H 0.062 ± 0.02 101.0 ± 3.8 1043-fluoro 0.225 ± 0.10  91.5 ± 8.4 105 4-fluoro 0.057 ± 0.02 101.9 ± 8.9106 2-chloro 0.298 ± 0.14  95.6 ± 10  107 3-chloro 0.126 ± 0.01  99.1 ±3.1 108 4-chloro 0.326 ± 0.09  90.6 ± 3.3 109 4-methyl 0.356 ± 0.12 84.1 ± 5.5 110 4-trifluoromethyl 0.553 ± 0.13  56.1 ± 5.4 111 4-mthoxy0.037 ± 0.01  96.2 ± 2.5 112 2,4-difluoro 0.044 ± 0.01  96.0 ± 6.2 1132,6-difluoro 0.049 ± 0.02  93.8 ± 5.0 114 2,3-difluoro 0.215 ± 0.06 72.8 ± 7.8 115 2-chloro-6-fluoro 0.060 ± 0.02  92.9 ± 4.1 1162,3,4-trifluoro 0.174 ± 0.07  69.2 ± 14.6 117 2,3,5,6-tetrafluoro 0.345± 0.06  59.4 ± 6.3 118 2-fluoro-3-methyl 0.035 ± 0.01  97.4 ± 6.9 1192-fluoro-4-methyl 0.108 ± 0.03  80.5 ± 5.8 1202-fluoro-4-trifluoromethyl >15  59.3 ± 18  121 2-fluoro-4-methoxy 0.225± 0.07  68.2 ± 7.9 ^(a)AC50 values were determined utilizing theluminescent pyruvate kinase-luciferase coupled assay and the datarepresents the results from three separate experiments. ^(b)Max Res.(Maximum Response) is % activity that represents % activation at 57 μMof compound. See Methods for normalization.

Additional compounds of Formula II and their properties are sets forthin Table 7. R¹⁴ to R¹⁶=H.

TABLE 7 KinaseGlo LDH Compound hPK, M2 hPK, M2 hPK, M2 hPK, M2 No. R¹¹R¹² R¹³ AC₅₀(μM)^(a) Max. Res.^(b) AC₅₀(μM)^(a) Max. Res.^(b) 147 Me Me6-fluoro 0.1834 83.26 0.2906 220.97 148 S(O)Me Me 3-methoxy 0.092 89.00nd nd 149 S(O)Me Me 3-amino 0.115 91.00 nd nd ^(a)Max Res. (MaximumResponse) is % activity that represents % activation at 57 μM ofcompound.

Following the examination of the core heterocycle and selectedappendages, a phenyl ring scan on the benzyl substituent was performed.The results suggest a less focused SAR for this moiety; however,selected trends did exist. For instance, bulky substituents weretypically not successful at the para position of the ring [for instance108 (AC₅₀=326±91 nM, maximum response=90.6%; LDH assay, AC₅₀=1,650±1,000nM and maximum response=191%), 110 (AC₅₀=553±134 nM, maximumresponse=56.1%; LDH assay, AC₅₀=2,200±830 nM and maximum response=77%)and 120 (AC₅₀>15 μM, maximum response <80%, in both assays))]. Electronwithdrawing substitutions were typically favored [for instance 112(AC₅₀=44±11 nM, maximum response=96.0%; LDH assay, AC₅₀=170±30 nM andmaximum response=217%), 113 (AC₅₀=49±18 nM, maximum response=93.8%; LDHassay, AC₅₀=140±10 nM and maximum response=240%)], however examples suchas the 4-methoxybenzyl analogue 111 were exceptions (AC₅₀=37±13 nM,maximum response=96.2%; LDH assay, AC₅₀=230±40 nM and maximumresponse=258%). Substitutions that confer favorable SAR were not alwaysadditive as is demonstrated by the 2-fluoro-4-methoxy analogue 121(AC₅₀=225±97 nM, maximum response=68.2%; LDH assay, AC₅₀=1,000±370 nMand maximum response=135±20%).

Mode of action. It was essential to establish the cooperative nature ofthese agents with the native substrates of PKM2. Given the allostericactivation of PKM2 by FBP, it was desirable to examine how the compoundsaffected the kinetics of PEP and ADP. In the absence of activator, hPKshows low affinity for PEP (K_(M) ˜1.5 mM). In the presence of 66 orFBP, the K_(M) for PEP decreased to 0.13±0.04 mM or 0.1±0.02 mM for thetwo activators, respectively. Comparison of the ADP titration in thepresence and absence of activators shows that these kinetics are notsignificantly affected (K_(M) for ADP ˜0.1 mM in either condition;V_(max) values within 20% of each other). Thus, NCGC00031955 (thesubstituted thieno[3,2-b]pyrrole[3,2-d]pyridazinone 66) activates PKM2by increasing the enzyme's affinity for PEP (FIG. 4A) and has littleeffect on ADP kinetics (FIG. 4B).

Selectivity of substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinones andselected analogues. With the SAR surrounding this chemotype establishedit was essential to consider the selectivity of these compounds versusPKM1, PKR and PKL. The N,N′-diarylsulfonamide chemotype presented in theaccompanying manuscript possessed a high degree of selectivity foractivation of PKM2. Gratifyingly, the substitutedthieno[3,2-b]pyrrole[3,2-d]pyridazinones presented here were equallyselective for PKM2 activation versus PKM1. Further, all analoguesexamined were inactive versus PKL and PKR (see PubChem AIDs listed inMethods). FIG. 6 details the selectivity of NCGC00031955 (66) versusPKM2, PKM1, PKR and PKL.

Example 4

This example illustrates some of the properties of a compound of FormulaIII:

TABLE 8 KinaseGlo LDH Compound hPK, M2 hPK, M2 hPK, M2 hPK, M2 No.Formula AC₅₀(μM) Max. Res.^(a) AC₅₀(μM) Max. Res.^(a) 150

  150 0.1634 84.04 0.1457 153.98 ^(a)Max Res. (Maximum Response) is %activity that represents % activation at 57 μM of compound.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A compound of Formula I:

wherein R¹ and R² are aryl or heteroaryl, optionally substituted withone or more substituents selected from the group consisting of C₁-C₁₀alkyl, C₃-C₆ alkylene, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ haloalkyl,C₁-C₁₀ dihaloalkyl, C₁-C₁₀ trihaloalkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, C₆-C₁₀ aryl, heterocyclyl, heteroaryl, heteroaryloxide,alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴, OCOR⁴, SCOR⁴, SOR⁴, SO₂R⁴,SO₂NR⁴R⁵, NO₂, B(OH)₂, CN, and halogen, and L is a linker comprising anamino group; or a pharmaceutically acceptable salt thereof; with theprovisos that R¹ and R² are not dimethoxyphenyl and that R¹ and R² arenot both 4-methylphenyl simultaneously.
 2. The compound or salt of claim1, wherein the compound of formula (I) is a compound of formula (Ia):

wherein n=1 to 3, R¹ and R² are aryl or heteroaryl, optionallysubstituted with one or more substituents selected from the groupconsisting of C₁-C₁₀ alkyl, C₃-C₆ alkylene, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₁-C₁₀ haloalkyl, C₁-C₁₀ dihaloalkyl, C₁-C₁₀ trihaloalkyl,C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀ aryl, heterocyclyl,heteroaryl, heteroaryloxide, alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴,OCOR⁴, SCOR⁴, SOR⁴, SO₂R⁴, SO₂NR⁴R⁵, NO₂, B(OH)₂, CN, and halogen, R³and R⁴ are independently selected from the group consisting of H, C₁-C₁₀alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, COR⁶, F, and CF₃, or, R³ and R⁴, taken together with thecarbon atom to which they are attached, form C═O, R⁵ and R⁷ to R¹⁰ areindependently H, C₁-C₁₀ alkyl, or F, R⁶ is C₁-C₁₀ alkyl or C₃-C₁₀cycloalkyl, or each of R⁷ and R⁸ and of R⁹ and R¹⁰, together form C═Oand X is CH or N, or a pharmaceutically acceptable salt thereof.
 3. Thecompound or salt according to claim 2, wherein R¹ and R² are phenylsubstituted with one or more substituents selected from the groupconsisting of C₁-C₁₀ alkyl, C₁-C₁₀ trihaloalkyl, heterocyclyl,heteroaryl, alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴, OCOR⁴, SCOR⁴, SOR⁴,SO₂R⁴, SO₂NR⁴R⁵, CN, and halogen, R³ and R⁴ are independently selectedfrom the group consisting of H, C₁-C₁₀ alkyl, and F, or, taken togetherwith the carbon atom to which they are attached, form C═O, and R⁵ and R⁷to R¹⁰ are independently H, C₁-C₁₀ alkyl, or F.
 4. (canceled)
 5. Thecompound or salt according to claim 3, wherein X is N and n is
 1. 6.(canceled)
 7. The compound or salt according to claim 2, wherein R¹ isselected from the group consisting of phenyl, 4-methylphenyl,2-methylphenyl, 2-fluorophenyl, 4-chlorophenyl, 4-fluorophenyl,4,2-difluorophenyl, 2,6-difluorophenyl, 2,4,5-trifluorophenyl,4-chloro-2-fluorophenyl, 3-chloro-2-fluorophenyl,4-trifluoromethylphenyl, 3-trifluoromethylphenyl,2,6-difluoro-4-trifluoromethylphenyl, 2,6-difluoro-4-methoxyphenyl,2,5-difluoro-4-propylphenyl, 2,6-difluoro-3-hydroxyphenyl,2,4-difluorophenyl, 4-bromo-2-fluorophenyl,2,6-difluoro-3-hydroxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl,4-cyanophenyl, 2-nitrophenyl, 2-pyridyl, 2-pyridyl-1-oxide, 2-(boronicacid)phenyl, 3-(boronic acid)phenyl, and 4-(boronic acid)phenyl. 8.(canceled)
 9. The compound or salt according to claim 2, wherein R² is6-(2,3-dihydrobenzo[b][1,4]dioxinyl),7-(3,4-dihydro-2H-benzo[b][1,4]dioxepinyl), 5-benzo[d][1,4]dioxinyl,7-(4-methyl-3,4-dihydro-2H-pyrido[3,2-b][1,4-oxazinyl), 2-naphthalenyl,6-(2,2-dimethylchromanyl), 5-(1-methyl-1H-indolyl),6-(2-methylbenzo[d]thiazolyl), or 4-methoxyphenyl.
 10. (canceled) 11.The compound or salt of claim 2, wherein the compound is of formula(Ia), X is N, n=1, and R³ to R¹⁰ is H, and R¹ and R² are as follows: R¹is 4-methoxyphenyl and R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹and R² are 6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ and R² are4-methoxyphenyl; R¹ is 4-cyanophenyl and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is 4-chlorophenyl and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is 4-fluorophenyl and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is 3-fluorophenyl and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is 2-fluorophenyl and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is 2,6-difluorophenyl and R²is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is 2,4,5-trifluorophenyland R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is2,6-difluoro-4-methoxyphenyl and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is 2,5-difluoro-3-propylphenyland R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is2,6-difluoro-3-hydroxyphenyl and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is 2,4-difluorophenyl and R²is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is phenyl and R² is6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is 3-(trifluoromethylphenyl)and R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is 3-methoxyphenyland R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is 4-methoxyphenyland R² is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is 2-pyridyl and R²is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is 2-pyridyl-1-oxide and R²is 6-(2,3-dihydro-benzo[b][1,4]dioxinyl); R¹ is 2,6-difluorophenyl andR² is 2,6-difluorophenyl; R¹ is 2,6-difluorophenyl and R² is7-(3,4-dihydro-2H-benzo[b][1,4]dioxepinyl); R¹ is 2,6-difluorophenyl andR² is 5-benzo[d][1,4]dioxinyl; R¹ is 2,6-difluorophenyl and R² is7-(4-methyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazinyl); R¹ is2,6-difluorophenyl and R² is 2-naphthalenyl; R¹ is 2,6-difluorophenyland R² is 6-(2,2-dimethylchromanyl); R¹ is 2,6-difluorophenyl and R² is5-(1-methyl-1H-indolyl); R¹ is 2,6-difluorophenyl and R² is6-(2-methylbenzo[d]thiazolyl); or R¹ is 2,6-difluorophenyl and R² is6-(2,3-dihydrobenzo[b][1,4]dioxinyl).
 12. (canceled)
 13. The compound orsalt of claim 2, wherein the compound is of formula (Ic):

wherein R³ to R¹⁰ are H or methyl, R³ to R⁶ and R⁹ and R¹⁰ are H ormethyl and R⁷ form C═O, or R³ to R⁸ are H or methyl and R⁹ and R¹⁰ takentogether with the carbon atom to which they are attached form C═O. 14.The compound or salt of claim 11, wherein (i) R⁵ is methyl and R³, R⁴,and R⁶ to R¹⁰ are H; (ii) R⁶ is methyl and R³ to R⁵ and R⁷ to R¹⁰ are H;(iii) R³ is methyl and R⁴ to R¹⁰ are H; (iv) R⁴ is methyl and R³ and R⁵to R¹⁰ are H; (v) R³ to R⁸ are H and R⁹ and R¹⁰ form C═O; or (vi) R³ toR⁶ and R⁷ and R⁸ are H and R⁷ and R⁸ taken together with the carbon atomto which they are attached form C═O.
 15. The compound or salt accordingto claim 2, wherein R¹ is selected from the group consisting of2-pyridyl, 2-pyridyl-N-oxide, 3-pyridyl, 3-pyridyl-N-oxide, 4-pyridyl,4-pyridyl-N-oxide, 2-pyrimidinyl, 2-pyrimidinyl-N-oxide, 4-pyrimidinyl,4-pyrimidinyl-N-oxide, 5-pyrimidinyl, 5-pyrimidinyl-N-oxide,2-pyrazinyl, and 2-pyrazinyl-N-oxide.
 16. The compound or salt of claim2, wherein X is CH.
 17. The compound or salt of claim 16, wherein nis
 1. 18. The compound or salt of claim 16, wherein R³, R⁴, and R⁵ areH.
 19. The compound or salt of claim 16, wherein R¹ is selected from thegroup consisting of 4-methylphenyl, 2-methylphenyl, 2-fluorophenyl,3-fluorophenyl, 4,2-difluorophenyl, 2,6-difluorophenyl,2,4,5-trifluorophenyl, 2,6-difluoro-4-trifluoromethylphenyl,4-chloro-2-fluoro, 3-chloro-2-fluoro, 4-trifluoromethylphenyl,4-bromo-2-fluorophenyl, 4-methoxyphenyl, and 2-nitrophenyl.
 20. Thecompound or salt according to claim 19, wherein R¹ is selected from thegroup consisting of 2,6-difluoro-4-trifluoromethylphenyl,2,6-difluorophenyl, and 4-methoxyphenyl.
 21. The compound or salt ofclaim 20, wherein R² is 3,4-ethylenedioxyphenyl.
 22. (canceled)
 23. Thecompound or salt of claim 1, wherein L is an alkylene diamino,cycloalkylamino amino, or cycloalkylamino alkylamino.
 24. The compoundor salt of claim 23, wherein L is N,N′-(ethane-1,2-diyl),N,N′-(propane-1,3-diyl), N,N′-(butane-1,4-diyl),N,N′-(pentane-1,5-diyl), N,N′-(hexane-1,6-diyl),N,N′-((trans)-cyclohexane-1,4-diyl), N,N′-((cis)-cyclohexane-1,4-diyl),


25. The compound or salt of claim 24, wherein R¹ is 2,6-difluorophenyland R² is 6-(2,3-dihydrobenzo[b][1,4]dioxinyl).
 26. A compound ofFormula II:

wherein: R¹¹ is selected from the group consisting of H, C₁-C₁₀ alkyl,C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl,C₆-C₁₀ aryl, OR¹⁷, SR¹⁷, SOR¹⁷, SO₂R¹⁷, NR¹⁷R¹⁸, NCOR¹⁷, SCOR¹⁷, COR¹⁷,OCOR¹⁷, B(OH)₂, NO₂, NHCOR¹⁷, CN, CHO, hydroxy C₁-C₁₀ alkyl, andhalogen, R¹² is selected from the group consisting of H, C₁-C₂ alkyl,allyl, C₃-C₁₀ cycloalkyl, NCOR¹⁴, and SO₂R¹⁴, R¹³ to R¹⁶ are selectedfrom the group consisting of H, C₁-C₁₀ alkyl, halo C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀aryl, heteroaryl, OR¹⁷, SR¹⁷, NR¹⁷R¹⁸, NCOR¹⁷, OCOR¹⁷, SCOR¹⁷, SOR¹⁷,SO₂R¹⁷, SO₂NR¹⁷R¹⁸, CF₃, and halogen, and R¹⁷ and R¹⁸ are independentlyselected from the group consisting of H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, and C₆-C₁₀ aryl,or a pharmaceutically acceptable salt thereof, with the proviso thatwhen R¹¹ is methyl, R¹² is methyl or allyl, and R¹⁴ to R¹⁶ are H, thenR¹³ is not methoxy or fluoro.
 27. The compound or salt according toclaim 26, wherein R¹¹ is selected from the group consisting of H, C₁-C₁₀alkyl, OR¹⁷, SR¹⁷, SOR¹⁷, SO₂R¹⁷, NR¹⁷R¹⁸, NCOR¹⁷, SCOR¹⁷, COR¹⁷,OCOR¹⁷, B(OH)₂, NO₂, NHCOR¹⁷, CN, CHO, hydroxy C₁-C₁₀ alkyl, andhalogen, R¹² is selected from the group consisting of H, methyl, NCOR¹⁴,and SO₂R¹⁴, R¹³ to R¹⁶ are selected from the group consisting of H,C₁-C₁₀ alkyl, OR¹⁷, SR¹⁷, NR¹⁷R¹⁸, NCOR¹⁷, OCOR¹⁷, SCOR¹⁷, SOR¹⁷,SO₂R¹⁷, SO₂NR¹⁷R¹⁸, CF₃, and halogen, and R¹⁷ and R¹⁸ are independentlyselected from the group consisting of H and C₁-C₁₀ alkyl.
 28. (canceled)29. (canceled)
 30. The compound or salt according to claim 27, whereinR¹¹ and R¹² are methyl, R¹³ is H, 2-fluoro, 3-fluoro, 4-fluoro,2-chloro, 3-chloro, 4-chloro, 4-CF₃, 4-methyl, or 4-methoxy, and R¹⁴ toR¹⁶ are H or halogen.
 31. The compound or salt according to claim 30,wherein R¹¹ and R¹² are methyl, and R¹³ and R¹⁴ are 2-fluoro and4-fluoro, 2-fluoro and 6-fluoro, 2-fluoro and 3-fluoro, 2-chloro and6-fluoro, 2-fluoro and 3-methyl, 2-fluoro and 4-methyl, 2-fluoro and4-CF₃, and 2-fluoro and 4-methoxy, and R¹⁵ and R¹⁶ are H.
 32. (canceled)33. (canceled)
 34. A pharmaceutical composition comprising a compound orsalt according to claim 1, and a pharmaceutically acceptable carrier.35. A method of treating a disease responsive to activation of humanPK-M2 comprising administering to a patient in need thereof atherapeutically effective amount of a compound of claim
 1. 36. Themethod of claim 35, wherein the compound is of formula Ia

wherein n=1 to 3, R¹ and R² are aryl, phenyl, or heteroaryl, optionallysubstituted with one or more substituents selected from the groupconsisting of C₁-C₁₀ alkyl, C₃-C₆ alkylene, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₁-C₁₀ haloalkyl, C₁-C₁₀ dihaloalkyl, C₁-C₁₀ trihaloalkyl,C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀ aryl, heterocyclyl,heteroaryl, heteroaryloxide, alkylenedioxy, OR⁴, SR⁴, NR⁴R⁵, NCOR⁴,OCOR⁴, SCOR⁴, SOR⁴, SO₂R⁴, SO₂NR⁴R⁵, NO₂, B(OH)₂, CN, and halogen, R³and R⁴ are independently selected from the group consisting of H, C₁-C₁₀alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, COR⁶, F, and CF₃, or, R³ and R⁴, taken together, form C═O,R⁵ and R⁷ to R¹⁰ are independently H, C₁-C₁₀ alkyl, or F, R⁶ is C₁-C₁₀alkyl or C₃-C₁₀ cycloalkyl, or each of R⁷ and R⁸ and of R⁹ and R¹⁰,together form C═O and X is CH or N.
 37. A method of treating a diseaseresponsive to activation of human PK-M2 comprising administering to apatient in need thereof a therapeutically effective amount of a compoundof claim
 26. 38. (canceled)
 39. A compound of formula III:

wherein R²¹ and R²² are aryl, substituted with one or more substituentsselected from the group consisting of C₁-C₁₀ alkyl, C₃-C₆ alkylene,C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ haloalkyl, dihaloalkyl, C₁-C₁₀trihaloalkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, C₆-C₁₀ aryl,heterocyclyl, heteroaryl, heteroaryloxide, alkylenedioxy, OR²³, SR²³,NR²³R²⁴, NCOR²³, OCOR²³, SCOR²³, SO₂R²³, SO₂NR²³R²⁴, NO₂, B(OH)₂, CN andhalogen, wherein R²³ and R²⁴ are independently H, C₁-C₁₀ alkyl, F,C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl,COR⁶, and CF₃, or a pharmaceutically acceptable salt thereof.
 40. Acompound of the formula:

or a pharmaceutically acceptable salt thereof.
 41. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and acompound or salt of claim
 40. 42. A method of treating a diseaseresponsive to activation of human PKM2 comprising administering to apatient in need thereof a therapeutically effective amount of a compoundor salt of claim
 40. 43. (canceled)
 44. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a compound or saltof claim
 26. 45. The method of claim 35, wherein the disease is cancer.46. The method of claim 45, wherein the cancer is selected from thegroup consisting of renal cancer, ovarian cancer, breast cancer, CNScancer, leukemia, prostate cancer, non-small cell lung cancer, coloncancer, and melanoma,
 47. The method of claim 46, wherein the cancer isselected from the group consisting of renal cancer, CNS cancer, breastcancer, and ovarian cancer.
 48. The method of claim 47, wherein thecancer is renal cancer.
 49. The method of claim 47, wherein the canceris CNS cancer.
 50. The method of claim 47, wherein the cancer is breastcancer.
 51. The method of claim 47, wherein the cancer is ovariancancer.
 52. The method of claim 37, wherein the disease is cancer. 53.The method of claim 52, wherein the cancer is selected from the groupconsisting of renal cancer, ovarian cancer, breast cancer, CNS cancer,leukemia, prostate cancer, non-small cell lung cancer, colon cancer, andmelanoma.
 54. The method of claim 53, wherein the cancer is selectedfrom the group consisting of renal cancer, CNS cancer, breast cancer,and ovarian cancer.
 55. The method of claim 54, wherein the cancer isrenal cancer.
 56. The method of claim 54, wherein the cancer is CNScancer.
 57. The method of claim 54, wherein the cancer is breast cancer.58. The method of claim 54, wherein the cancer is ovarian cancer. 59.The method of claim 42, wherein the disease is cancer.
 60. The method ofclaim 59, wherein the cancer is selected from the group consisting ofrenal cancer, ovarian cancer, breast cancer, CNS cancer, leukemia,prostate cancer, non-small cell lung cancer, colon cancer, and melanoma.61. The method of claim 60, wherein the cancer is selected from thegroup consisting of renal cancer, CNS cancer, breast cancer, and ovariancancer.
 62. The method of claim 61, wherein the cancer is renal cancer.63. The method of claim 61, wherein the cancer is CNS cancer.
 64. Themethod of claim 61, wherein the cancer is breast cancer.
 65. The methodof claim 61, wherein the cancer is ovarian cancer.
 66. The method ofclaim 35, wherein the disease is anemia.
 67. The method of claim 37,wherein the disease is anemia.
 68. The method of claim 42, wherein thedisease is anemia.